Figure 6A GST pulldown showing that dL ( 3 ) mbt , the Drosophila homolog of L3MBTL3 , directly interacts with Su ( H ) , the Drosophila homolog of RBPJ . In vitro transcribed and translated dL ( 3 ) mbt or dNotch ICD ( dNotch ICD fragment containing the RAM domain and ANK repeats ) , as positive control , was incubated with bacterially purified GST - Su ( H ) or GST alone pre - bound to GSH beads . Proteins were resolved via SDS - PAGE and signals were acquired via X - ray exposure . The experiment was independently replicated four times . C Snapshot showing the co - localization of dL ( 3 ) mbt and Su ( H ) at the dNotch ( N ) locus . D In the wing imaginal disc , dL ( 3 ) mbt overexpression in the dorso - ventral ( D - V ) boundary results in the downregulation of the Notch target gene cut . Wing discs expressing UAS - GFP ( top panels ) or UAS - HA - dL ( 3 ) mbt ; UAS - GFP ( bottom panels ) under the vg - Gal4 driver at 25 ° C were stained for cut and HA . GFP marks the vg - Gal4 expression domain . Insets below each panel show a closer view of the D - V boundary with yellow arrows marking the regions where HA - dL ( 3 ) mbt is expressed and cut is downregulated . At least 20 discs for each genotype were analyzed . Representative images are shown . Scale bars : 100μm . E The vg - Gal4 - driven HA - dL ( 3 ) mbt overexpression causes a serrated wing ( wing notching ) phenotype . Flies expressing either UAS - GFP or UAS - HA - dL ( 3 ) mbt ; UAS - GFP under vg - Gal4 were reared to adulthood at 25 ° C . P values were estimated by comparing the proportions via a two - proportion Z - test . Scale bars : 200μm . F Functional interaction between lag - 1 / RBPJ and lin - 61 / L3MBTL3 during C . elegans vulva development . Proportion of animals ( n ≥ 100 ) displaying a protruding vulva ( Pvl ) phenotype after RNAi treatment for two generations . Worms were grown at 25 ° C . Shown are means ± s . d of duplicate experiments . EV : Empty Vector control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A GST pulldown showing that dL(3)mbt, the Drosophila homolog of L3MBTL3, directly interacts with Su(H), the Drosophila homolog of RBPJ. In vitro transcribed and translated dL(3)mbt or dNotch ICD (dNotch ICD fragment containing the RAM domain and ANK repeats), as positive control, was incubated with bacterially purified GST-Su(H) or GST alone pre-bound to GSH beads. Proteins were resolved via SDS-PAGE and signals were acquired via X-ray exposure. The experiment was independently replicated four times.C Snapshot showing the co-localization of dL(3)mbt and Su(H) at the dNotch (N) locus.D In the wing imaginal disc, dL(3)mbt overexpression in the dorso-ventral (D-V) boundary results in the downregulation of the Notch target gene cut. Wing discs expressing UAS-GFP (top panels) or UAS-HA-dL(3)mbt;UAS-GFP (bottom panels) under the vg-Gal4 driver at 25°C were stained for cut and HA. GFP marks the vg-Gal4 expression domain. Insets below each panel show a closer view of the D-V boundary with yellow arrows marking the regions where HA-dL(3)mbt is expressed and cut is downregulated. At least 20 discs for each genotype were analyzed. Representative images are shown. Scale bars: 100μm.E The vg-Gal4-driven HA-dL(3)mbt overexpression causes a serrated wing (wing notching) phenotype. Flies expressing either UAS-GFP or UAS-HA-dL(3)mbt;UAS-GFP under vg-Gal4 were reared to adulthood at 25°C. P values were estimated by comparing the proportions via a two-proportion Z-test. Scale bars: 200μm.F Functional interaction between lag-1/RBPJ and lin-61/L3MBTL3 during C. elegans vulva development. Proportion of animals (n ≥100) displaying a protruding vulva (Pvl) phenotype after RNAi treatment for two generations. Worms were grown at 25°C. Shown are means ±s.d of duplicate experiments. EV: Empty Vector control.


Figure 1A The distribution of the GFP - tagged Hxt2 changes during the cell division . In small and medium budded cells ( white arrowheads ) , Hxt2 is mainly present in the membrane of the mother cell . In large budded cells , Hxt2 is distributed over the entire plasma membrane ( yellow arrowheads ) . B HXT2 mRNA localization correlate with that of the protein . FISH with an HXT2 - specific probe . Early in the cell cycle the mRNA is predominantly found in the mother cell ( white arrowheads ) while it is equally distributed after meta - to - anaphase transition ( MAT ) ( yellow arrowheads ) . C Quantification of ( B ) . The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively . D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton . Cells were either treated for 15 min with 30 mg / ml latrunculin A or with 30 mg / ml benomyl . White arrowheads point to small - budded and yellow arrowheads to large budded cells . E Quantification of ( D ) of three in dependent experiments with at least 50 cells each . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A The distribution of the GFP-tagged Hxt2 changes during the cell division. In small and medium budded cells (white arrowheads), Hxt2 is mainly present in the membrane of the mother cell. In large budded cells, Hxt2 is distributed over the entire plasma membrane (yellow arrowheads).B HXT2 mRNA localization correlate with that of the protein. FISH with an HXT2-specific probe. Early in the cell cycle the mRNA is predominantly found in the mother cell (white arrowheads) while it is equally distributed after meta-to-anaphase transition (MAT) (yellow arrowheads). C Quantification of (B). The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells. E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.


Figure 2A , Inhibiting translation by either applying Cycloheximide ( CHX ) or Verrucarin A ( VerruA ) leads to the retention of HXT2 mRNA in the mother cell after MAT . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX . FISH of CHX or VerruA - treated cells . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX . C CHX treatment leads to increased transcript stability . Quantitative PCR of HXT2 mRNA in CHX - treated cells in comparison to prt1 - 1 mutant cells . The data represents 3 independent experiments and the standard deviation is given . D Translation of HXT2 mRNA is more active late in the cell cycle . The HXT2 mRNA level bound to either di - / trisomes and polysomes ( ≥ 4 ribosomes ) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1 / S or M - phase followed by qPCR . At G1 / S similar levels of the HXT2 mRNA were found in the different ribosome fractions , whereas in M - phase more mRNA was bound to polysomes , suggesting a more active translation in M - phase . E The localization of HXT2 mRNA is independent of the Signal Recognition Particle ( SRP ) . Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given.D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase.E The localization of HXT2 mRNA is independent of the Signal Recognition Particle (SRP). Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA.


Figure 3A Deletion of SCP160 , ASC1 , or BFR1 increases HXT2 mRNA signal in the bud . FISH experiment with deletions in various RNA binding proteins . B The effect of ∆ scp160 on HXT2 mRNA localization is independent of its increased ploidy . Scp160 truncations that lack either the last two ( ∆ C2 ) or the last four ( ∆ C4 ) KH domains or a Tet - off SCP160 construct , in which expression is blocked by the addition of doxycycline ( Doxy ) D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud . Treatment of cells with forskolin ( + Forsko ) or using a ∆ bcy1 strain , causes HXT2 mRNA to be bud - enriched . E HXT2 mRNA enrichment upon refeeding is Ras2 - dependent .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O

Figure 3A Deletion of SCP160, ASC1, or BFR1 increases HXT2 mRNA signal in the bud. FISH experiment with deletions in various RNA binding proteins.B The effect of ∆scp160 on HXT2 mRNA localization is independent of its increased ploidy. Scp160 truncations that lack either the last two (∆C2) or the last four (∆C4) KH domains or a Tet-off SCP160 construct, in which expression is blocked by the addition of doxycycline (Doxy)D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud. Treatment of cells with forskolin (+Forsko) or using a ∆bcy1 strain, causes HXT2 mRNA to be bud-enriched.E HXT2 mRNA enrichment upon refeeding is Ras2-dependent.


Figure 4A Table of the glucose affinity of the four most important hexose transporters . B FISH of HXT1 , 3 and 4 under glucose rich conditions . White arrowheads depict cells before , yellow arrowheads cells after MAT . C Quantification of ( B ) . D Hyperactive PKA promotes enrichment of all HXTs after MAT . E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation . F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding . Daughters and mothers were separated through elutriation . qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions . G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2 - GFP transporter levels at the plasma membrane of the daughter cells . Daughter cells were enriched through elutriation . Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment . Western blot analysis confirms the higher amount of Hxt2 - GFP transporter protein present in daughter cells under glucose - shift

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Table of the glucose affinity of the four most important hexose transporters. B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT. C Quantification of (B). D Hyperactive PKA promotes enrichment of all HXTs after MAT.E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation.F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding. Daughters and mothers were separated through elutriation. qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions.G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2-GFP transporter levels at the plasma membrane of the daughter cells. Daughter cells were enriched through elutriation. Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment. Western blot analysis confirms the higher amount of Hxt2-GFP transporter protein present in daughter cells under glucose-shift conditions


Figure 5A RNA degradation is not essential for asymmetric HXT2 mRNA localization . FISH of wild - type and ∆ xrn1 cells . B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud . Cells were treated upon glucose addition with either 120 mg / ml benomyl for 15 min , 30 mg / ml latrunculin A for 30 min or 200 µM CK - 666 for 30 min . HXT2 mRNA was detect by FISH . C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function . HXT2 mRNA FISH with ∆ bim1 and ∆ kar9 strains . Arrows point to a nucleus with HXT2 signal . D Quantification of the daughter cell enrichment from data displayed in ( C ) . E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in ( C ) . Percentage of cells with asymmetric HXT2 mRNA staining is displayed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A RNA degradation is not essential for asymmetric HXT2 mRNA localization. FISH of wild-type and ∆xrn1 cells.B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud. Cells were treated upon glucose addition with either 120 mg/ml benomyl for 15 min, 30 mg/ml latrunculin A for 30 min or 200 µM CK-666 for 30 min. HXT2 mRNA was detect by FISH.C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function. HXT2 mRNA FISH with ∆bim1 and ∆kar9 strains. Arrows point to a nucleus with HXT2 signal. D Quantification of the daughter cell enrichment from data displayed in (C). E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in (C). Percentage of cells with asymmetric HXT2 mRNA staining is displayed.


Figure 6A Deletion of NUP2 and MLP1 / 2 reduced HXT2 mRNA enrichment in the bud . FISH under glucose shift conditions with strains in which different NPC components were deleted . B Quantification of ( A ) . C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift . FISH - IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84 - HA . White arrows point to HXT2 mRNA signals on the nuclear envelope . D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift . Stills of time lapse movies . HXT2 mRNA was visualized using the MS2 - GFP system . Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus . Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus . Cyan : Nup84 - mCherry ; white arrowhead : HXT2 mRNA - MS2 + MCP - GFP .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted. B Quantification of (A). C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope.D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift. Stills of time lapse movies. HXT2 mRNA was visualized using the MS2-GFP system. Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus. Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus. Cyan: Nup84-mCherry; white arrowhead: HXT2 mRNA-MS2 + MCP-GFP.


Figure 7HXT2 only strains grow faster than the other HXT only strain . Growth was assessed 2 - 3 days after incubation at 30 ° C . D Quantification of competition assay . qPCR with primers against either HXT1 , 2 , 3 or 4 was performed . HXT gene abundance was normalized against actin . E Strains that lack HXT2 grow slower when coming from quiescence compared to WT . F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96 % of cells independently of the cell cycle . Arrows point to examples of HXT2 - U1A mRNA in the bud early and late in the cell cycle . G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2 . H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers . Cells were re - fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7HXT2 only strains grow faster than the other HXT only strain. Growth was assessed 2-3 days after incubation at 30°C.D Quantification of competition assay. qPCR with primers against either HXT1, 2, 3 or 4 was performed. HXT gene abundance was normalized against actin.E Strains that lack HXT2 grow slower when coming from quiescence compared to WT.F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96% of cells independently of the cell cycle. Arrows point to examples of HXT2-U1A mRNA in the bud early and late in the cell cycle.G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2.H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers. Cells were re-fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points.


Figure 1A . Cryo - sections of human organoids were analyzed for the presence of enterocytes ( E - cad ) , Goblet cells ( Muc - 2 ) and tight junctions ( ZO - 1 ) by indirect immunofluorescence . Nuclei are stained with DAPI . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test . B . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . 16hpi organoids were frozen , cryo - sectioned , and HAstV1 infected cells were visualized by indirect immunofluorescence ( HAstV1 ( red ) , nuclei ( DAPI , blue ) . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test . C . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells ( green ) were visualized by indirect immunofluorescence . Apical and basolateral membranes were immunostained for actin ( magenta ) and Laminin ( red ) respectively . Nuclei are stained with DAPI ( blue ) . Scale bar is 20 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test . E . Human intestinal organoids were infected with HAstV1 . At indicated time post - infection the increase in viral copy number was determined by q - RT - PCR . F . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . At 24 hpi the presence of IFNλ in the media was tested by ELISA . Dotted line indicates detection limit of the assay . G . Same as E but for the induced levels of either type I IFN ( IFNβ1 ) or type III IFN ( IFNλ ) . H . Human intestinal organoids were pre - treated for 24h with 2000IU / mL of IFNβ1 or 300ng / mL of IFNλ1 - 3 . Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q - RT - PCR .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.B. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.C. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red) respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.E. Human intestinal organoids were infected with HAstV1. At indicated time post-infection the increase in viral copy number was determined by q-RT-PCR.F. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay.G. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ).H. Human intestinal organoids were pre-treated for 24h with 2000IU/mL of IFNβ1 or 300ng/mL of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q-RT-PCR.


Figure 3J - M . Percentage of stem cells ( OLFM4 ) goblet cell ( FGCBP ) , mature enterocytes ( APOA4 ) and cells of the enterocyte lineage ( FGCBP ) from the total organoid cells population ( black bar ) in mock - treated organoids and after HAstV1 infection . The percentage of infected cells ( red bars ) and bystander cells ( blue bars ) within each cell type is shown . The data was obtained from multiplex in situ RNA hybridization in 2D organoids . n = 10 , mean ± SEM . Statistics show comparison of infected cells between mock - treated , 4h pi and 16h pi organoids . An Ordinary one - way ANOVA and Tukey ' s multiple comparisons test was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.


Figure 5A . One image per condition was used to show the multiplex in situ RNA FISH . A representative region of the images was chosen and cropped in order to have a zoom - in of a specific area for better visualization . Shown are the enterocyte lineage marker FABP6 ( green ) , HAstV1 infection ( white ) , and CCL2 gene expression ( red ) of mock - treated and infected organoids . DAPI is in blue . White arrows show co - localization of FABP6 and CCL2 with HAstV1 signal . Scale bar 100µm . B - D . Fluorescence intensity ( arbitrary units ) of innate immune marker expression in stem cells ( OLFM4 positive ) , goblet cells ( FCGBP positive ) , enterocyte lineage cells ( FABP6 positive ) , and mature enterocytes ( APOA4 positive ) . Bystander cells are in blue and infected cells in red . 10 - 90 percentile box plots , each dot represents one cell ( mock N = 2274 , 4h N = 3682 , 16h N = 1510 . C . mock N = 2693 , 4h N = 3706 , 16h N = 1921 . D . mock N = 2274 , 4h N = 3706 , 16h N = 1514 . ) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages . Ordinary one - way ANOVA and Tukey ' s multiple comparisons test were used . Grey statistics show the comparison between infected and bystander cells within each cell lineage . Unpaired t - test with Welch ' s correction was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. One image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show co-localization of FABP6 and CCL2 with HAstV1 signal. Scale bar 100µm.B-D. Fluorescence intensity (arbitrary units) of innate immune marker expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4 positive). Bystander cells are in blue and infected cells in red. 10-90 percentile box plots, each dot represents one cell (mock N=2274, 4h N=3682, 16h N=1510. C. mock N=2693, 4h N=3706, 16h N=1921. D. mock N=2274, 4h N=3706, 16h N=1514.) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Grey statistics show the comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001


Figure 1 ( A ) Overexpression of RNF26 promoted polyubiquitination of MITA . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with a control or RNF26 expression plasmid ( 0 . 5 , 1 , 2 µg ) . Twenty - four hours after transfection , cells were subjected to immunoprecipitation ( IP ) under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - HA ( upper panel ) , anti - Flag ( middle panel ) and anti - RNF26 ( bottom panel ) . Ub , ubiquitin . ( B ) RNF26 - mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26 . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with RNF26 or its mutants ( 1 µg each ) . IP under denatured conditions and ubiquitination assay was performed as described in ( A ) . ( C ) UbcH5 mediated polyubiquitination of MITA by RNF26 . The RNF26 and MITA proteins were obtained by in vitro transcription and translation , then incubated with biotin - Ub , E1 and the indicated E2s . Polyubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . ( D ) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro . MITA , RNF26 and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and MITA were incubated with RNF26 or its mutants , followed by ubiquitination and immunoblot analysis as described in ( C ) . All experiments were repeated for at least three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Overexpression of RNF26 promoted polyubiquitination of MITA. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with a control or RNF26 expression plasmid (0.5, 1, 2 µg). Twenty-four hours after transfection, cells were subjected to immunoprecipitation (IP) under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-HA (upper panel), anti-Flag (middle panel) and anti-RNF26 (bottom panel). Ub, ubiquitin.(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.


Figure 2 ( A ) RNF26 interacted with MITA . The 293 cells ( 5 × 106 ) were transfected with the indicated plasmids ( 5 µg each ) . Twenty - four hours later , cells were lysed and cell lysates were immunoprecipitated with anti - Flag or control IgG . The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti - HA or anti - Flag . ( B ) RNF26 was physiologically associated with MITA . THP - 1 cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins . NC , negative control . ( C ) RNF26 is localized to the ER and mitochondria . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Twenty - four hours after transfection , the cells were stained with Mito - Tracker Red for 15 minutes or left untreated . The cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . D ) Subcellular distribution of RNF26 . The 293 cells ( 5 × 107 ) were infected with SeV for the indicated time points or left uninfected . Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins . ( E ) RNF26 and MITA were colocalized to the ER . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Eighteen hours after transfection , cells were infected with SeV or HSV - 1 for 6 hours or left uninfected , followed by ER - Tracker Blue / White staining for 30 minutes . Cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . All experiments were repeated for at least three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) RNF26 interacted with MITA. The 293 cells (5×106) were transfected with the indicated plasmids (5 µg each). Twenty-four hours later, cells were lysed and cell lysates were immunoprecipitated with anti-Flag or control IgG. The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti-HA or anti-Flag.(B) RNF26 was physiologically associated with MITA. THP-1 cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins. NC, negative control.(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.D) Subcellular distribution of RNF26. The 293 cells (5×107) were infected with SeV for the indicated time points or left uninfected. Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins.(E) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.


Figure 3 ( A ) RNF26 mediated polyubiquitination of MITA at K150 . The 293 cells ( 5 × 106 ) were transfected with HA - Ub ( 1 µg ) and RNF26 ( 1 µg ) together with Flag - MITA or the indicated mutants ( 5 µg each ) . Twenty - four hours after transfection , cells were subjected IP under denatured conditions with anti - Flag and immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . B ) RNF26 targeted MITA for polyubiquitination at K150 in vitro . RNF26 , MITA and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and RNF26 were incubated with MITA or its mutants . The ubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . All experiments were repeated for at least three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.


Figure 4 ( A and B ) RNF26 targeted MITA for K11 - linked polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Twenty - four hours after transfection , cells were subjected for IP under denatured conditions with limited amount of anti - Flag ( 0 . 5 µg ) so that equal amount of Flag - MITA is pulled down . The immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . Ub - AKR , all lysine residues of ubiquitin were mutated to arginine . ( C ) Effects of RNF26 - RNAi plasmids on the expression of RNF26 . In the upper panels , the 293 cells ( 1 × 106 ) were transfected with the expression plasmids for RNF26 - Flag ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with the indicated RNAi plasmids ( 1 µg each ) . Twenty - four hours after transfection , whole cell lysates were analyzed by immunoblots with anti - Flag or anti - HA . In the lower panels , 293 cells were transduced with a control or RNF26 - RNAi by retrovirus mediated gene transfer . Cells ( 1 × 106 ) were lysed and whole cell lysates were analyzed by immunoblots with anti - RNF26 or anti - β - actin . ( D ) Immunoblot analysis of Flag - tagged ubiquitin expression in THP - 1 cells stably transfected with Flag - Ub - K11O plasmid . Whole cell lysates of THP - 1 - Flag - Ub - K11O - RNF26 - RNAi and control cells ( 1 × 106 ) were analyzed by immunoblots with antibodies against the indicated proteins . RNF26 - RNAi # 1 was used here and in the following experiments if not noted . ( E and F ) Effects of RNF26 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . In ( E ) , THP - 1 - Flag - Ub - K11O - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti - MITA . The immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panels ) or anti - MITA ( lower panels ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins . In ( F ) , 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with a control or RNF26 - RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , puromycin ( 1 µg / mL ) was added into the culture medium . The cells were selected for twenty - four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in ( E ) . All experiments were repeated for at least three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A and B) RNF26 targeted MITA for K11-linked polyubiquitination. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Twenty-four hours after transfection, cells were subjected for IP under denatured conditions with limited amount of anti-Flag (0.5 µg) so that equal amount of Flag-MITA is pulled down. The immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. Ub-AKR, all lysine residues of ubiquitin were mutated to arginine.(C) Effects of RNF26-RNAi plasmids on the expression of RNF26. In the upper panels, the 293 cells (1×106) were transfected with the expression plasmids for RNF26-Flag (0.5 µg) and HA-β-actin (0.1 µg) together with the indicated RNAi plasmids (1 µg each). Twenty-four hours after transfection, whole cell lysates were analyzed by immunoblots with anti-Flag or anti-HA. In the lower panels, 293 cells were transduced with a control or RNF26-RNAi by retrovirus mediated gene transfer. Cells (1×106) were lysed and whole cell lysates were analyzed by immunoblots with anti-RNF26 or anti-β-actin.(D) Immunoblot analysis of Flag-tagged ubiquitin expression in THP-1 cells stably transfected with Flag-Ub-K11O plasmid. Whole cell lysates of THP-1-Flag-Ub-K11O-RNF26-RNAi and control cells (1×106) were analyzed by immunoblots with antibodies against the indicated proteins. RNF26-RNAi #1 was used here and in the following experiments if not noted.(E and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.


Figure 5A ) Effects of RNF26 knockdown on virus - induced polyubiquitination of endogenous MITA . The THP - 1 - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Ub ( K48 ) ( upper panel ) , anti - Ub ( K63 ) ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins . ( B ) Effects of RNF5 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . The 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with the indicated RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , the cells were selected with puromycin ( 1 µg / mL ) for twenty - four hours and infected with SeV for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies . ( C ) RNF26 and RNF5 competed with each other on MITA polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with MITA ( 5 µg ) , Flag - Ub - K48O and HA - Ub - K11O ( 1 µg each ) together with indicated amount of RNF26 and RNF5 . Twenty - four hours after transfection , cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panel ) , anti - HA ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins . ( D ) Effects of RNF26 knockdown on virus - triggered MITA degradation . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins ( upper immunoblots ) . The relative protein levels of MITA in reference to β - actin were analyzed by Quantity One program and data shown are mean ± S . D . of three independent experiments ( lower histographs ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Effects of RNF26 knockdown on virus-induced polyubiquitination of endogenous MITA. The THP-1-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Ub(K48) (upper panel), anti-Ub(K63) (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.(B) Effects of RNF5 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. The 293-HA-Ub-K11O cells (2×107) were transfected with the indicated RNAi plasmid (10 µg each). Twelve hours after transfection, the cells were selected with puromycin (1 µg/mL) for twenty-four hours and infected with SeV for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies.(C) RNF26 and RNF5 competed with each other on MITA polyubiquitination. The 293 cells (5×106) were transfected with MITA (5 µg), Flag-Ub-K48O and HA-Ub-K11O (1 µg each) together with indicated amount of RNF26 and RNF5. Twenty-four hours after transfection, cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panel), anti-HA (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.(D) Effects of RNF26 knockdown on virus-triggered MITA degradation. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins (upper immunoblots). The relative protein levels of MITA in reference to β-actin were analyzed by Quantity One program and data shown are mean ± S.D. of three independent experiments (lower histographs).


Figure 6 ( A ) Effects of RNF26 knockdown on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and the indicated RNAi plasmid ( 0 . 5 µg each ) . Thirty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed . ( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B ) ( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B ) or ELISA analysis ( C ( D ) Effects of RNF26 knockdown on virus - triggered induction of IFNB1 gene in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with VSV , EMCV or ECTV for the indicated time points or left uninfected before quantitative real - time PCR analysis was performed as in ( B ) . ( E and F ) Effects of RNF26 knockdown on virus - triggered induction of TNFα in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( E ) and ELISA analysis ( F ) . ( G ) Effects of RNF26 knockdown on virus - triggered phosphorylation of TBK1 , IRF3 and IκBα . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected , whole cell lysates were analyzed by immunoblots with anti - p - TBK1 , anti - TBK1 , anti - p - IRF3 , anti - IRF3 , anti - p - IκBα , anti - IκBα , anti - RNF26 or anti - β - actin as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B)(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C(D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B).(E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F).(G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


Figure 7A ) Effects of RNF26 knockdown on IRF3 level . In the left panel , whole cell lysates of THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were analyzed by immunoblots with the indicated antibodies . In the right panel , cells ( 1 × 106 ) were subjected to quantitative real - time PCR analysis . B ) Effects of RNF26 on IRF3 stability . The 293 cells ( 1 × 106 ) were transfected with HA - IRF3 ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with RNF26 or its mutant ( 0 . 3 µg each ) . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 . ( C ) Effects of RNF26 and its mutant on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and RNF26 or its mutant ( 0 . 1 µg each ) . Twenty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed . ( D ) Effects of RNF26 on IRF3 ubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - IRF3 ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment . The cell lysates were subjected to IP under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panels ) or anti - Flag ( lower panels ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . ( E ) Effects of inhibitors on RNF26 - mediated destabilization of IRF3 . The 293 cells ( 1 × 106 ) were transfected with the indicated plasmids as described in ( B ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) , NH4Cl ( 25 mM ) or MG132 ( 100 µM ) was added into culture medium for four hours . Whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 . ( F ) Effects of ATG12 knockdown on RNF26 - mediated destabilization of IRF3 . The 293 - ATG12 - RNAi or control cells ( 1 × 106 ) were transfected with the indicated plasmids . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA , anti - RNF26 or anti - ATG12 as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×106) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×106) were subjected to quantitative real-time PCR analysis.B) Effects of RNF26 on IRF3 stability. The 293 cells (1×106) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.(C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.(F) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×106) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


Figure 1 ( A , B ) Expression of UVRAG under cell stress . HL60 and HCT116 cells were treated with doxorubicin ( 1 μg / ml ) , cisplatin ( 10 μm ) and UV irradiation ( 5 min after 50 mJ / cm2 ) for the indicated time , and then apoptosis , autophagy and fluorescence intensity of UVRAG was assayed . Scale bar , 10 μm . ( C ) In parallel , the total protein extracts were used for western blot analysis . ( D , E ) Pharmacological inhibition ( for example , 3 ‐ methyladenine ( 3 ‐ MA ) , 10 mM ) or genetic deletion ( for example , ATG5 − / − ) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin ( Doxo ) and UV irradiation ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; C ‐ PARP , cleaved poly ‐ ADP ribose polymerase ; LC3 , light chain 3 ; mRNA , messenger RNA ; PI , propidium iodide ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance ‐ associated gene .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 (A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm. (C) In parallel, the total protein extracts were used for western blot analysis.(D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.


Figure 2 ( A - C ) After transfection with UVRAG shRNA or control shRNA for 48 h , HL60 ( A ) , HCT116 ( B ) and HeLa ( C ) cells were treated with doxorubicin ( Doxo , 1 μg / ml ) , cisplatin ( Cis , 10 μm ) and UV irradiation ( 5 min after 50 mJ / cm2 ) for 12 h or starvation ( HBSS ) for 3 h , and then apoptosis and autophagy were assayed ( n = 3 , * P0 . 05 ) . ( D ) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A ( 10 μg / ml ) and E64D ( 10 μg / ml ) after doxorubicin ( 1 μg / ml ) treatment for 12 h . * P0 . 05 compared with control shRNA group . ( E ) After transfection with UVRAG shRNA or control shRNA for 48 h , ATG5 + / + and ATG5 − / − murine embryonic fibroblasts ( MEFs ) were treated with doxorubicin ( 1 μg / ml ) , with or without Z ‐ VAD ‐ FMK ( 20 μM ) for 12 h , and then apoptosis was assayed ( n = 3 , * P0 . 05 ) . ( F ) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control ( shControl ) or UVRAG ( shUVRAG ) ‐ specific shRNA and treated with doxorubicin ( 10 mg / kg ) beginning at day 7 . Tumours were measured twice weekly , and volumes were calculated for 21 days ( n = 10 , * P0 . 05 ; shControl + Doxo compared with shUVRAG + Doxo ) . ( G ) On day 21 in the experiments described in ( F ) , apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting , respectively ( n = 10 , * P0 . 05 ) . BAX , BCL2 ‐ associated X protein ; HBSS , Hank ' s Buffered Salt Solution ; LC3 , light chain 3 ; shRNA , short ‐ hairpin RNA ; TUNEL , terminal deoxytransferase uridine triphosphate nick end labelling ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05).(D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. *P0.05 compared with control shRNA group.(E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5+/+ and ATG5−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z‐VAD‐FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, *P0.05).(F) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)‐specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, *P0.05; shControl+Doxo compared with shUVRAG+Doxo).(G) On day 21 in the experiments described in (F), apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting, respectively (n=10, *P0.05). BAX, BCL2‐associated X protein; HBSS, Hank's Buffered Salt Solution; LC3, light chain 3; shRNA, short‐hairpin RNA; TUNEL, terminal deoxytransferase uridine triphosphate nick end labelling; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 3 ( A ) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid , and apoptosis was assayed ( n = 3 , * P0 . 05 ) . ( B ) In parallel , the mitochondrial membrane potential , cytochrome c release from mitochondria , and caspase ‐ 3 and ‐ 9 activation in HL60 cells were analysed ( n = 3 , * P0 . 05 ) . ( C ) After transfection with UVRAG vector or control vector for 48 h , cells were transected or treated with BAX ( 1 μg ) , BAD ( 1 μg ) , BID ( 1 μg ) , FAS antibody ( 10 μg / ml ) or TRAIL ( 100 ng / ml ) for 24 h , then cell apoptosis was analysed ( n = 3 , * P0 . 05 ) . ( D ) Knockdown of UVRAG in BAX − / − HCT116 cells restored the sensitivity to UV treatment ‐ induced apoptosis ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; BAX , BCL2 ‐ associated X protein ; shRNA , short ‐ hairpin RNA ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, *P0.05).(B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase‐3 and ‐9 activation in HL60 cells were analysed (n=3, *P0.05).(C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, *P0.05).(D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment‐induced apoptosis (n=3, *P0.05). AU, arbitrary units; BAX, BCL2‐associated X protein; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 4 ( A , B ) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co ‐ immunoprecipitation ( RIPA or CHAPS buffer ) or western blotting . ( C ) Proteins in the pellet after GST pull ‐ down assays in HCT116 cell lysate were analysed by SDS - PAGE and immunodetected with UVRAG or GST antibodies . ( D ) UVRAG C2 domain deletion terminates BAX binding . HCT116 cells were transfected with Flag ‐ UVRAG or its mutants ( ΔC2 , ΔCCD ) , and then assayed for protein expression levels as indicated by IP or western blot . ( E ) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin ( Doxo , 1 μg / ml ) , UV irradiation ( 5 min after 50 mJ / cm2 ) or transfected with BAX ( 1 μg ) for 24 h . BAX , BCL2 ‐ associated X protein ; CCD , coiled ‐ coil domain ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate ; GST , glutathione ‐ S ‐ transferase ; IB , immunoblotting ; IgG , immunoglobulin G ; IP , immunoprecipitation ; RIPA , radioimmunoprecipitation assay ; SDS - PAGE , SDS - polyacrylamide gel electrophoresis ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co‐immunoprecipitation (RIPA or CHAPS buffer) or western blotting.(C) Proteins in the pellet after GST pull‐down assays in HCT116 cell lysate were analysed by SDS-PAGE and immunodetected with UVRAG or GST antibodies.(D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag‐UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot.(E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2‐associated X protein; CCD, coiled‐coil domain; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate; GST, glutathione‐S‐transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 5 UVRAG inhibits mitochondrial translocation of BAX . After transfection with indicated shRNA or cDNA for 48 h , HL60 cells were treated with doxorubicin ( Doxo ) and UV irradiation for 12 h , and then BAX in cytosol ( Cyt ) and mitochondria ( Mit ) was assayed by western blotting ( A - C ) and activation of BAX ( D - F ) was assayed by western blotting after IP using BAX monoclonal antibody ( clone 6A7 ) * P0 . 05 . AU , arbitrary units ; BAX , BCL2 ‐ associated X protein ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate buffer ; Cyt c , cytochrome c ; IP , immunoprecipitation ; shRNA , short ‐ hairpin RNA ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A-C) and activation of BAX (D-F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P0.05. AU, arbitrary units; BAX, BCL2‐associated X protein; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 2A . Average ribosome occupancy and their respective codon bias - derived occupancy scores across the CDS of transcript ( in 25 bins ) . Ribosome occupancy for 16 , 423 transcripts and their respective codon bias - derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin . B . Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias - derived occupancy . Correlations obtained from the ribosome occupancy and scrambled codon bias - derived occupancy served as the control . A Kolmogorov - Smirnov test was performed between the codon bias - derived occupancy and the control group . Data information : error bars represent the 95 % confidence intervals . C . Comparison of average transcript translation efficiencies ( TEs ) across their respective GC3 - content ranges . Number of transcripts within each gene optimality range is indicated above their respective points . Data information : error bars represent the 95 % confidence intervals .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Average ribosome occupancy and their respective codon bias-derived occupancy scores across the CDS of transcript (in 25 bins). Ribosome occupancy for 16,423 transcripts and their respective codon bias-derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin. B. Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias-derived occupancy. Correlations obtained from the ribosome occupancy and scrambled codon bias-derived occupancy served as the control. A Kolmogorov-Smirnov test was performed between the codon bias-derived occupancy and the control group. Data information: error bars represent the 95% confidence intervals.C. Comparison of average transcript translation efficiencies (TEs) across their respective GC3-content ranges. Number of transcripts within each gene optimality range is indicated above their respective points. Data information: error bars represent the 95% confidence intervals.


Figure 3A . HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( left ) as well as IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( right ) , post - doxycycline addition . Data information : data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets . B . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells transfected with either empty plasmids , plasmids bearing REL - OPT or REL - WT . The immunoblot is representative of 3 independent experiments . ACTB is shown as the loading controls . C . ELISA of secreted IL6 concentrations of IL6 - OPT , IL6 - WT and IL6 - DE from HEK293T cells transfected with plasmids bearing IL6 - OPT , IL6 - WT and IL6 - DE . Data information : In ( C ) , the data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A one - way ANOVA with Tukey ' s multiple comparisons was performed between samples where , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . D . Fold changes of REL - OPT and REL - WT transcript levels ( top ) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions ( below ) . Data represents the mean ± SD for 3 biological replicates . Data information data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.B. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells transfected with either empty plasmids, plasmids bearing REL-OPT or REL-WT. The immunoblot is representative of 3 independent experiments. ACTB is shown as the loading controls.C. ELISA of secreted IL6 concentrations of IL6-OPT, IL6-WT and IL6-DE from HEK293T cells transfected with plasmids bearing IL6-OPT, IL6-WT and IL6-DE. Data information: In (C), the data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A one-way ANOVA with Tukey's multiple comparisons was performed between samples where, p<0.01 (**) and p<0.001 (***).D. Fold changes of REL-OPT and REL-WT transcript levels (top) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions (below). Data represents the mean ± SD for 3 biological replicates. Data information data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


Figure 4HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( B ) , under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( C ) under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( D ) , under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of REL - OPT , REL - OPT ( + 1 Frameshift ) and REL - WT transcripts ( E ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT , IL6 - DE and IL6 - OPT ( + 1 Frameshift ) transcripts ( F ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (B), under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (C) under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (D), under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of REL-OPT, REL-OPT (+1 Frameshift) and REL-WT transcripts (E) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT, IL6-DE and IL6-OPT (+1 Frameshift) transcripts (F) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


Figure 5Volcano plots showing the enrichment of RBPs which bind to REL - WT relative to REL - OPT transcripts ( A ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 . Volcano plots showing the enrichment of RBPs which bind to , IL6 - DE relative to IL6 - WT transcripts ( B ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 . Volcano plots showing the enrichment of RBPs which bind to IL6 - WT relative to IL6 - OPT transcripts ( C ) . Data information : , vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Volcano plots showing the enrichment of RBPs which bind to REL-WT relative to REL-OPT transcripts (A) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.Volcano plots showing the enrichment of RBPs which bind to , IL6-DE relative to IL6-WT transcripts (B) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.Volcano plots showing the enrichment of RBPs which bind to IL6-WT relative to IL6-OPT transcripts (C). Data information: , vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.