Figure 2A Dose response curves of ID188 ( p27 - 29 ) and ID211 ( p26 - 28 ) organoids treated with FOLFIRINOX over three days . ATP was measured with CellTiter - Glo assays . Shown is the mean ± SD of three independent experiments . C Plot of the delta AUC in ID188 ( p5 + 27 ) versus ID211 ( p9 + 11 ) cells over 415 drugs . Dashed lines demark the threshold of delta AUC > 0 . 3 or < - 0 . 3 . D Heatmap of the screening hits as defined in C . AUC is color coded . E - J Dose response curves of ID188 and ID211 organoids ( ID188 p27 - 29 , ID211 p26 - 28 ) or 2D lines ( ID188 p5 + 29 , ID211 p9 + 13 ) treated with ( E and F ) : Cobimetinib or ( H and I ) : Biminetinib . ( G ) Cobimetinib and ( J ) Biminetinib : GI50 values . Shown is the mean ± SD of three independent experiments . K Illustration of a cohort of patients ( n = 15 ) with available primary 2D PDAC cell lines . This cohort includes the ID188 ( pre - FFX ( FOLFIRINOX ) , red ) and ID211 ( post - FFX , blue ) lines . These lines were screened for sensitivity towards three MEKi . L 15 primary human PDAC 2D cell lines were screened for MEKi ( as indicated ) sensitivity . The determined AUC was variance scaled and the z - scores are depicted . The ID188 and ID211 identity is color coded .

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Figure 2A Dose response curves of ID188 (p27-29) and ID211 (p26-28) organoids treated with FOLFIRINOX over three days. ATP was measured with CellTiter-Glo assays. Shown is the mean ± SD of three independent experiments.C Plot of the delta AUC in ID188 (p5+27) versus ID211 (p9+11) cells over 415 drugs. Dashed lines demark the threshold of delta AUC >0.3 or <-0.3. D Heatmap of the screening hits as defined in C. AUC is color coded.E - J Dose response curves of ID188 and ID211 organoids (ID188 p27-29, ID211 p26-28) or 2D lines (ID188 p5+29, ID211 p9+13) treated with (E and F): Cobimetinib or (H and I): Biminetinib. (G) Cobimetinib and (J) Biminetinib: GI50 values. Shown is the mean ± SD of three independent experiments.K Illustration of a cohort of patients (n=15) with available primary 2D PDAC cell lines. This cohort includes the ID188 (pre-FFX (FOLFIRINOX), red) and ID211 (post-FFX, blue) lines. These lines were screened for sensitivity towards three MEKi. L 15 primary human PDAC 2D cell lines were screened for MEKi (as indicated) sensitivity. The determined AUC was variance scaled and the z-scores are depicted. The ID188 and ID211 identity is color coded.


Figure 1Treatment of HeLa cells with DFP ( 1mM for 24 h ) reduces the conjugation of SUMO - 2 / 3 ( n = 5 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change global SUMO - 1 - ylation ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to increased levels of SENP3 ( n = 8 , biological replicates ; * * p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change levels of SENP5 ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) . DFP treatment increases SENP3 stability . DFP ( 1mM for 24 h ) - treated HeLa cells were treated with cycloheximide ( CHX ; 50μg / ml ) for the indicated durations in a chase experiment . Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated .

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Figure 1Treatment of HeLa cells with DFP (1mM for 24 h) reduces the conjugation of SUMO-2/3 (n=5, biological replicates; **, p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change global SUMO-1-ylation (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).  Treatment of HeLa cells with DFP (1mM for 24 h) leads to increased levels of SENP3 (n=8, biological replicates; **p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change levels of SENP5 (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).  DFP treatment increases SENP3 stability. DFP (1mM for 24 h)-treated HeLa cells were treated with cycloheximide (CHX; 50μg/ml) for the indicated durations in a chase experiment. Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated.


Figure 2Treatment of HeLa cells with DFP ( 1mM for 24 h ) decreases levels of CHIP ( n = 8 , biological replicates ; * * * p < 0 . 001 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to decreased levels of CHIP mRNA ( n = 3 , biological replicates ; * p < 0 . 05 ; unpaired t - test ) . Treatment of DFP ( 1mM for 24 h ) - treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP . Histogram shows relative levels of cellular CHIP upon four different conditions ( n = 3 , biological replicates ; * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; repeated measures analysis of variance followed by Tukey ' s multiple comparisons test ) . CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells . Nsi or CHIPi ( Nsi , non - specific siRNA ; CHIPi , CHIP siRNA ; concentration , 20 nM ) was transfected into HeLa cells for 48 h . Lysate samples were blotted as indicated . CHIP knockdown prevents the DFP - induced increase in SENP3 levels . Nsi , SENP3i or CHIPi ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; CHIPi , CHIP siRNA ; siRNA concentration , 20nM ) was transfected into HeLa cells for 48 h . Two days post - transfection the cells were treated with DFP for a further 24 h .

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Figure 2Treatment of HeLa cells with DFP (1mM for 24 h) decreases levels of CHIP (n=8, biological replicates; ***p<0.001; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of CHIP mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).  Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP. Histogram shows relative levels of cellular CHIP upon four different conditions (n=3, biological replicates; ****p<0.0001; *** p<0.001; repeated measures analysis of variance followed by Tukey's multiple comparisons test).CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells. Nsi or CHIPi (Nsi, non-specific siRNA; CHIPi, CHIP siRNA; concentration, 20 nM) was transfected into HeLa cells for 48 h. Lysate samples were blotted as indicated.   CHIP knockdown prevents the DFP-induced increase in SENP3 levels. Nsi, SENP3i or CHIPi (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; CHIPi, CHIP siRNA; siRNA concentration, 20nM) was transfected into HeLa cells for 48 h. Two days post-transfection the cells were treated with DFP for a further 24 h.


Figure 3SENP3 knockdown abolishes the DFP - induced increase in LC3 - II in HeLa cells . Nsi or SENP3i ( I ) was transfected into HeLa cells . Two days post - transfection the cells were treated with DFP for a further 24 h . Whole cell lysate samples were blotted as indicated ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; siRNA concentration , 20nM ; DFP , 1mM for 24 h ; values normalized to the control value ; n = 3 , biological replicates ; * * * , p < 0 . 001 ; N . S . , not significant , p > 0 . 05 ; paired t - test ) . Nsi or SENP3i ( II ) was transfected into HeLa cells with stable expression of mito - Keima grown on 6 - well plates . Two days post - transfection the cells were treated with DFP ( 1mM ; 24 h ) . Cells transfected with Nsi and treated with CQ ( 50μM ; 24 h ) in the absence or presence of DFP ( 1mM ; 24 h ) were included as a negative control for mitophagy induction . The images show overlaid fluorescence and transmitted light images of unfixed , live cells obtained using an EVOS - fl inverted LED fluorescence microscope ( Red : mitophagic puncta ; Scale bar , 100 μm ) . Histogram shows the percentages of mitophagic cells under 6 different conditions ( n = 3 , biological replicates ; * * * * , p < 0 . 0001 ; Ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test ) . SENP3 knockdown abolishes DFP - induced mitophagic autolysosomes . Nsi or SENP3i ( I ) together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , and were analyzed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Magenta : SENP3 ; Scale bar , 10 μm ; n = 28 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - test ) .

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Figure 3SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test).SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).


Figure 4Fis1 knockdown abolishes DFP - induced LC3 - II . Fis1 siRNA was introduced into HeLa cells for 48 h , and the cells were treated with DFP for further 24 h ( Nsi , non - specific siRNA ; Fis1i , Fis1 siRNA ; siRNA concentration , 20nM ; DFP , 1mM ) . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 3 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) . Fis1 knockout abolishes DFP - induced LC3 - II . Fis1 + / + or Fis1 - / - cells were treated with DFP for 24 h . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 6 , biological replicates ; * , p < 0 . 05 ; paired t - test ) . Fis1 knockdown prevents DFP - induced mitophagic autolysosomes . Nsi or Fis1i together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , which were analysed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Blue : Fis1 ; Scale bar , 10 μm ; n = 25 cells per condition from three individual experiments ; * * * p < 0 . 001 ; unpaired - t test ) .

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Figure 4Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test).Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test).Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).


Figure 5Flag - Fis1 is deSUMOylated by SENP3 . SENP3 knockdown enhances Fis1 SUMOylation . Nsi or SENP3i was transfected into HEK293 cells expressing Flag - Fis1 , His - SUMO - 2 and Ubc9 for 48 h . His - pulldown and lysate samples were detected by immunoblotting for Flag , SENP3 or β - actin . Values are presented as mean ± SEM ( n = 3 , biological replicates ; * p < 0 . 05 ; paired - t test ) .

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Figure 5Flag-Fis1 is deSUMOylated by SENP3. SENP3 knockdown enhances Fis1 SUMOylation. Nsi or SENP3i was transfected into HEK293 cells expressing Flag-Fis1, His-SUMO-2 and Ubc9 for 48 h. His-pulldown and lysate samples were detected by immunoblotting for Flag, SENP3 or β-actin. Values are presented as mean ± SEM (n=3, biological replicates; * p<0.05; paired-t test).


Figure 6The SUMOylation status of Fis1 influences its mitochondrial localization . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG was expressed in HeLa cells for 48 h . Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated ( the upper panel ) . Histograms ( the right panel ) show normalized levels of Flag - Fis1 WT or Flag - Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction ( Value are presented as mean ± SEM and are normalized to the control value ; n = 7 , biological replicates ; * , p < 0 . 05 ; paired t - test ) . The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum ( ER ) . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG along with GFP - Cb5 as an ER marker were expressed in HeLa cells for 48 h . Immunocytochemistry was then performed ( Green : ER labelled by GFP - Cb5 ; Blue : different Flag - Fis1 forms ; Scale bar , 10 μm )

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Figure 6The SUMOylation status of Fis1 influences its mitochondrial localization. Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG was expressed in HeLa cells for 48 h. Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated (the upper panel). Histograms (the right panel) show normalized levels of Flag-Fis1 WT or Flag-Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction (Value are presented as mean ± SEM and are normalized to the control value; n=7, biological replicates; *, p<0.05; paired t-test).The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum (ER). Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG along with GFP-Cb5 as an ER marker were expressed in HeLa cells for 48 h. Immunocytochemistry was then performed (Green: ER labelled by GFP-Cb5; Blue: different Flag-Fis1 forms; Scale bar, 10 μm)


Figure 7Expressing a SUMOylation - deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP - induced LC3 - II induction . pcDNA3 , Flag - Fis1 WT or Flag - Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ( I ) ; siRNA concentration , 20nM ) , and the cells were treated with DFP for a further 24 h ( DFP , 1mM ) . Lysate samples were blotted as indicated . SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP - Fis1 K149R in the presence of DFP . CFP - Fis1 WT or CFP - Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h , and the cells were treated with DFP for a further 24 h ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Cyan / blue : CFP - Fis1 ; Magenta , SENP3 ; Scale bar , 10 μm ) ; Values are presented as mean ± SEM ( n = 21 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - test ) . Knockdown of SENP3 was further confirmed by immunoblotting ( the lower right panel ) .

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Figure 7Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated.SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).


Figure 1B . BrdU - sensitized U - 2 - OS cells stably expressing GFP - NLS - CDKL5 were pre - incubated with DMSO ( mock ) , olaparib ( 5 μM ) , talazoparib ( 50 nM ) or PDD00017273 ( 0 . 3 μM ) for 1 h prior to micro - irradiation and live - imaging at the indicated times post - irradiation . One of three independent experiments is shown . Scale bar is 10 μmE . BrdU - sensitized parental or PARP1Δ / Δ , PARP2Δ / Δ , PARP1 / 2Δ / Δ U - 2 - OS cells transiently expressing GFP NLS - CDKL5 were subjected to 355 nm line micro - irradiation followed by time lapse imaging . One of two independent experiments is shown . Scale bar is 10 μmG . Cells subjected to the workflow in F . were detergent - extracted and fixed before staining with anti - GFP or fibrillarin ( nucleoli ) . Scale bar is 10 μm H . Quantification of the detergent - insoluble GFP - NLS - CDKL5 signal ( minus nucleolar signal ) . The mean ± SD from three biological experiments is shown . Statistical significance was assessed by one - way - ANOVA - test . Asterisks * * indicate P - value of < 0 . 01 ; ns - not significant .

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Figure 1B. BrdU-sensitized U-2-OS cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273 (0.3 μM) for 1 h prior to micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μmE. BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ, PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μmG. Cells subjected to the workflow in F. were detergent-extracted and fixed before staining with anti-GFP or fibrillarin (nucleoli). Scale bar is 10 μm H. Quantification of the detergent-insoluble GFP-NLS-CDKL5 signal (minus nucleolar signal). The mean ± SD from three biological experiments is shown. Statistical significance was assessed by one-way-ANOVA-test. Asterisks ** indicate P-value of <0.01; ns - not significant.


Figure 2B . BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS , the GFP - NLS - CDKL5 deletion mutants shown in A , or full length ( FL ) GFP - NLS - CDKL5 were subjected to line micro - irradiation ( 355 nm ) and time lapse imaging . Three independent experiments were performed , and one representative experiment is shown . Scale bar is 10 μmD . Same as in B except that the GFP - NLS tagged CDKL5 fragments indicated were examined . Scale bar is 10 μmF . Recombinant fragments of CDKL5 fused to GST ( 1 . 2 , 2 . 5 , 5 , 10 µg ) , or GST , were dot - blotted on nitrocellulose membrane and then incubated with synthetic PAR . PAR binding was detected by far western blotting . APLF was used as positive control . One of three independent experiments is shown ( G - H ) . U - 2 - OS ( Flp - In T - Rex ) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min . Extracts were subjected to immunoprecipitation with antibodies against CDKL5 ( G ) or PAR ( H ) ( or non - specific IgG as control ) . Precipitates , and input lysates , were analyzed by western blotting using the indicated antibodies . One of two independent experiments is

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Figure 2B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL) GFP-NLS-CDKL5 were subjected to line micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μmD. Same as in B except that the GFP-NLS tagged CDKL5 fragments indicated were examined. Scale bar is 10 μmF. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on nitrocellulose membrane and then incubated with synthetic PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown(G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting using the indicated antibodies. One of two independent experiments is shown


Figure 3A . ( Top ) BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro - irradiation ( 405 nm ) . ( Bottom ) Quantitation of spot intensities . Data represent the mean ± SEM of two independent experiments ; > 50 micro - irradiated cells per point . Scale bar is 10 μmBrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 , mCherry - XRCC1 or mCherry - FAN1 were pre - incubated with indicated transcription inhibitors prior to line micro - irradiation ( 355 nm ) and time lapse imaging . One of three independent experiments is shown . Scale bar is 10 μmE . Same as B - D . except that BrdU - sensitized cells stably expressing GFP - NLS - CDKL5 were also pre - incubated with olaparib as control . Cells were subjected to line micro - irradiation , fixed and then subjected to indirect immunofluorescence using antibodies against GFP , PAR and γH2AX . Scale bar is 10 μmF . Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging . Scale bar is 10

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Figure 3A. (Top) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. Scale bar is 10 μmBrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μmE. Same as B-D. except that BrdU-sensitized cells stably expressing GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μmF. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm


Figure 5B . Boxplots showing VSN - normalised intensity of phospho - peptides corresponding to EP400 pSer729 , ELOA pSer311and TTDN1 pSer40 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . In all cases the data was derived from 3 biological replicates . C . Boxplots of the VSN - adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG - EP400 , FLAG - ELOA and FLAG - TTDN1 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . Datapoints further removed than the whiskers are plotted individually . The experiment was conducted using three biological replicates within each respective group , each TMT channel represents a single biological replicate .

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Figure 5B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates.C. Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400, FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT channel represents a single biological replicate.


Figure 6A . HEK293 cells were co - transfected with CDKL5 ( wild type " WT " or kinase - dead " KD " K42R mutant ) fused to an NLS , and FLAG - ELOA ( wild type " WT " or a S311A mutant " SA " ) . Anti - FLAG precipitates or cell extracts were probed with the antibodies indicated . One of three independent experiments is shown . B . Same as A . showing a range of pathogenic ( red ) and benign ( blue ) CDKL5 variants . ( C - E ) . Wild type ( WT ) , CDKL5 - disrupted ( CDKL5Δ / Δ ) or siRNA - transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks . Quantification of ELOA - pSer311 signal at the laser tracks is shown . Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated ( n ) . For simplicity , only intensities greater than zero are shown . Statistical significance was assessed by one - way - ANOVA - test or unpaired t test with Welch ' s correction . Asterisks * * * * indicate P - values of < 0 . 0001 . Scale bar is 10

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Figure 6A. HEK293 cells were co-transfected with CDKL5 (wild type "WT" or kinase-dead "KD" K42R mutant) fused to an NLS, and FLAG-ELOA (wild type "WT" or a S311A mutant "SA"). Anti-FLAG precipitates or cell extracts were probed with the antibodies indicated. One of three independent experiments is shown. B. Same as A. showing a range of pathogenic (red) and benign (blue) CDKL5 variants. (C-E). Wild type (WT), CDKL5-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm


Figure 7D . ( Left ) Quantification of transcription in U - 2 - OS 263 IFII reporter cells from experiment in B . > 150 cells were analysed per condition per experiment . The mean ± SD from four independent experiments is shown . ( Right ) Quantitative RT - PCR analysis of YFP - MS2 mRNA in U - 2 - OS 263 reporter cells . Data represent mean ± SD in different biological replicates as indicated ( n ) . Statistical significance for all the data was assessed by two - way - ANOVA - test , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant .

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Figure 7D. (Left) Quantification of transcription in U-2-OS 263 IFII reporter cells from experiment in B. >150 cells were analysed per condition per experiment. The mean ± SD from four independent experiments is shown. (Right) Quantitative RT-PCR analysis of YFP-MS2 mRNA in U-2-OS 263 reporter cells. Data represent mean ± SD in different biological replicates as indicated (n). Statistical significance for all the data was assessed by two-way-ANOVA-test, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant.


Figure 8B , C . Quantitative PCR with reverse transcription ( qRT - PCR ) analysis of SLCO5a1 ( B ) and RYR2 expression levels ( C ) ( left panels ) U - 2 - OS HA - ER - I - PpoI cells depleted of CDKL5 transiently transfected with FLAG - tagged CDKL5 wild type ( WT ) or a K42R - mutated kinase dead ( KD ) mutant , or empty vector , at the times indicated after inducing I - PpoI . The mean ± SD from two qPCR replicates of two independent experiments is shown . Statistical significance for all the data was assessed by two - way - ANOVA - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant . I - PpoI - mediated cutting efficiency in the relevant gene is shown in the right - hand

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Figure 8B, C. Quantitative PCR with reverse transcription (qRT-PCR) analysis of SLCO5a1 (B) and RYR2 expression levels (C) (left panels) U-2-OS HA-ER-I-PpoI cells depleted of CDKL5 transiently transfected with FLAG-tagged CDKL5 wild type (WT) or a K42R-mutated kinase dead (KD) mutant, or empty vector, at the times indicated after inducing I-PpoI. The mean ± SD from two qPCR replicates of two independent experiments is shown. Statistical significance for all the data was assessed by two-way-ANOVA-test *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant. I-PpoI-mediated cutting efficiency in the relevant gene is shown in the right-hand panel


Figure 2A . Heatmap summary of Aft1 induction experiment showing all genes that change with | log2 ( fold - change ) | > 1 . 5 .

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Figure 2A. Heatmap summary of Aft1 induction experiment showing all genes that change with |log2(fold-change)| > 1.5.


Figure 1 ( D ) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp ( without ATG ) to the sequence from 5 ' cap of pri - miR - 31 to ATG site or which mutated to ATT of the sORF . Cells were selected by G418 for 2 days . Analysis of the GFP expression by confocal microscopy or flow cytometry , scale bar 100 μm . ( E ) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP - N1 - miPEP31 or pEGFP - N1 , the red arrow indicate predicted size of miPEP31 - GFP band . ( F ) Flow - fish to detect the localization of pri - miR - 31 . The location of pri - miR - 31 was analyzed by confocal microscopy . Scale bar 5 μm . ( G ) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31 . Values are expressed as fold changes relative to controls and normalized to GAPDH , the red arrow indicate predicted size of miPEP31 . ( H ) Immunoblot analysis of spleen , lymph node and thymus from WT or miR - 31 - / - mice with miPEP31 antibody , the red arrow indicate predicted size of miPEP31 .

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Figure 1(D) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp (without ATG) to the sequence from 5' cap of pri-miR-31 to ATG site or which mutated to ATT of the sORF. Cells were selected by G418 for 2 days. Analysis of the GFP expression by confocal microscopy or flow cytometry, scale bar 100 μm.(E) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP-N1-miPEP31 or pEGFP-N1, the red arrow indicate predicted size of miPEP31-GFP band.(F) Flow-fish to detect the localization of pri-miR-31. The location of pri-miR-31 was analyzed by confocal microscopy. Scale bar 5 μm.(G) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31. Values are expressed as fold changes relative to controls and normalized to GAPDH, the red arrow indicate predicted size of miPEP31.(H) Immunoblot analysis of spleen, lymph node and thymus from WT or miR-31-/- mice with miPEP31 antibody, the red arrow indicate predicted size of miPEP31.


Figure 3 ( A ) CD4 + T cells were activated with anti - CD3 / 28 and treated with different concentration of FAM - miPEP31 for 24 hours , the fluorescein was detected by Flow Cytometry . ( B ) Confocal Microscopy was used to analyze the fluorescence of FAM - miPEP31 in anti - CD3 / 28 activated CD4 + T cells treated with 10 μM FAM - miPEP31 . Scale bar 5 μm . Anti - CD3 / 28 activated CD4 + T cells were cultured with 10 μM miPEP31 for 24 hours . ( C ) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates . Anti - CD3 / 28 activated CD4 + T cells were cultured with 10 μM miPEP31 for 24 hours . ( D ) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4 + T and Treg cells treated with or without 10 μM miPEP31 . Data in ( D ) are presented as mean ± SEM of four biological replicates . ns , not significant ( CD4 + T versus Treg ) , unpaired two - tailed Student ' s t - test . . ( E ) Naïve CD4 + T cells were isolated from WT C57BL / 6 mice , and activated by anti - CD3 / 28 for 48 hours . Subsequently , FAM - labeled miPEP31 was added . Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4 + T cells . Scale bar 7 μm . ( F , G ) C57BL / 6 mice were injected intravenously with 100 μg FAM - labeled miPEP31 . Splenocytes were isolated and stained for CD4 and DAPI . Imaging Flow Cytometry analysis of miPEP31 in CD4 + T cells ( F ) and non - CD4 splenocytes ( G ) , scale bar 10 μm .

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Figure 3(A) CD4+ T cells were activated with anti-CD3/28 and treated with different concentration of FAM-miPEP31 for 24 hours, the fluorescein was detected by Flow Cytometry.(B) Confocal Microscopy was used to analyze the fluorescence of FAM-miPEP31 in anti-CD3/28 activated CD4+ T cells treated with 10 μM FAM-miPEP31. Scale bar 5 μm.Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (C) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates.Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (D) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4+ T and Treg cells treated with or without 10 μM miPEP31. Data in (D) are presented as mean ± SEM of four biological replicates. ns, not significant (CD4+ T versus Treg), unpaired two-tailed Student's t-test..(E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, FAM-labeled miPEP31 was added. Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4+ T cells. Scale bar 7 μm.(F, G) C57BL/6 mice were injected intravenously with 100 μg FAM-labeled miPEP31. Splenocytes were isolated and stained for CD4 and DAPI. Imaging Flow Cytometry analysis of miPEP31 in CD4+ T cells (F) and non-CD4 splenocytes (G) , scale bar 10 μm.


Figure 4 ( D , E ) Naïve CD4 + T cells were isolated from WT C57BL / 6 mice , and activated by anti - CD3 / 28 for 48 hours . Subsequently , 10 μM FAM - labeled miPEP31 was added for 20 min at 4 ℃ or 37 ℃ . Confocal Microscopy analysis of the fluorescence . Scale bar 10 μm . The localization of miPEP31 with DAPI was shown in ( E ) . Data are representative nuclear co - localization of miPEP31 in four representative images from four biological experiments . ( mean ± SEM ) . * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t - test .

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Figure 4(D, E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, 10 μM FAM-labeled miPEP31 was added for 20 min at 4℃ or 37℃. Confocal Microscopy analysis of the fluorescence. Scale bar 10 μm. The localization of miPEP31 with DAPI was shown in (E). Data are representative nuclear co-localization of miPEP31 in four representative images from four biological experiments. (mean ± SEM). ****p < 0.0001, unpaired two-tailed Student's t-test.


Figure 5scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization . ( D , E ) H & E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization . Histopathology scores of inflammation and demyelination were shown in ( E ) . Scale bars , 70 μm . Data in ( C , E ) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each . * * * *

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Figure 5scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization. (D, E) H&E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization. Histopathology scores of inflammation and demyelination were shown in (E). Scale bars, 70 μm. Data in (C, E) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each. ****


Figure 1IL - 1β concentrations in supernatants of macrophages , unstimulated ( control ) ( B ) or stimulated with SP / N ( C ) or LPS / N ( D ) . E . SARS - CoV - 2 spike ELISA of the corresponding plasma : ( EC50 titer ) of samples collected at T0 ( n = 35 individuals ) , T1 ( n = 28 individuals ) or T2 ( n = 44 individuals ) . F . Immunofluorescence microscopy images of ASC specks ( green ) in T2 macrophages . Nuclei were stained with DAPI ( blue ) . Scale bar indicates 20 µm . Arrow indicates ASC speck formation with magnified illustration in the right corner of the image . G , H . ASC specks were quantified microscopically at T0 ( G ) ( n = 7 individuals ) and T2 ( H ) ( n = 8 individuals ) upon indicated stimuli . I . Cleaved gasdermin D ( lysate ) and cleaved IL - 1β ( supernatant ) detected by Western Blot in macrophages stimulated with LPS / N or SP / N at T0 and T2 . J , K . Cell death quantification of macrophages by flow cytometry in unstimulated or SP / N stimulated cells at T0 ( n = 5 individuals ) ( J ) and T2 ( n = 5 individuals ) ( K ) using the viability dye Zombie UV . LPS / N was used as positive control . L . LDH release of macrophages ( n = 6 individuals ) stimulated with SP / N at T2 compared to unstimulated cells . M . IL - 1β concentrations of MCC950 treated T2 macrophage ( n = 44 individuals ) stimulation with SP / N .

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Figure 1IL-1β concentrations in supernatants of macrophages, unstimulated (control) (B) or stimulated with SP/N (C) or LPS/N (D).E. SARS-CoV-2 spike ELISA of the corresponding plasma: (EC50 titer) of samples collected at T0 (n = 35 individuals), T1 (n = 28 individuals) or T2 (n = 44 individuals).F. Immunofluorescence microscopy images of ASC specks (green) in T2 macrophages. Nuclei were stained with DAPI (blue). Scale bar indicates 20 µm. Arrow indicates ASC speck formation with magnified illustration in the right corner of the image.G, H. ASC specks were quantified microscopically at T0 (G) (n = 7 individuals) and T2 (H) (n = 8 individuals) upon indicated stimuli.I. Cleaved gasdermin D (lysate) and cleaved IL-1β (supernatant) detected by Western Blot in macrophages stimulated with LPS/N or SP/N at T0 and T2.J, K. Cell death quantification of macrophages by flow cytometry in unstimulated or SP/N stimulated cells at T0 (n = 5 individuals) (J) and T2 (n = 5 individuals) (K) using the viability dye Zombie UV. LPS/N was used as positive control.L. LDH release of macrophages (n = 6 individuals) stimulated with SP/N at T2 compared to unstimulated cells. M. IL-1β concentrations of MCC950 treated T2 macrophage (n = 44 individuals) stimulation with SP/N.


Figure 2B . Volcano plot showing DEGs in the same samples . Negative log10 padj - values are plotted against the log2 fold - change . Selected genes are labeled . C , D . CLEC4D ( C ) and CLEC4E ( D ) surface expression on patient - derived macrophages prior ( n = 5 individuals ) or post ( n = 5 individuals ) vaccination were quantified by flow cytometry . Graphs indicate the mean fluorescence intensity ( MFI ) . E , F . SP or LPS as a control PAMP were incubated with affinity purified CLRs ( ratio : 10 vs . 1 for SP ) for 1h and then added to primary T2 macrophages ( n = 5 individuals ) . Subsequently , IL - 1β was quantified in the supernatants . ( E ) shows absolute values for SP ; ( F ) shows the fold change calculated with the corresponding unstimulated control for SP and LPS . G . Monocytes were isolated prior ( n = 5 individuals ) or after vaccination ( n = 5 individuals ) and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin . H . Monocytes were isolated prior ( T0 ; n = 35 individuals ) or after vaccination [ 2 weeks after 1st ( T1 ; n = 28 individuals ) , 2 weeks ( T2 ; n = 38 individuals ) and 10 weeks ( T3 ; n = 33 individuals ) after 2nd vaccination ] and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages , stimulated with the SP S2 subunit SP are shown .

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Figure 2B. Volcano plot showing DEGs in the same samples. Negative log10 padj-values are plotted against the log2 fold-change. Selected genes are labeled.C, D. CLEC4D (C) and CLEC4E (D) surface expression on patient-derived macrophages prior (n = 5 individuals) or post (n = 5 individuals) vaccination were quantified by flow cytometry. Graphs indicate the mean fluorescence intensity (MFI).E, F. SP or LPS as a control PAMP were incubated with affinity purified CLRs (ratio: 10 vs. 1 for SP) for 1h and then added to primary T2 macrophages (n = 5 individuals). Subsequently, IL-1β was quantified in the supernatants. (E) shows absolute values for SP; (F) shows the fold change calculated with the corresponding unstimulated control for SP and LPS.G. Monocytes were isolated prior (n = 5 individuals) or after vaccination (n = 5 individuals) and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin. H. Monocytes were isolated prior (T0; n = 35 individuals) or after vaccination [2 weeks after 1st (T1; n = 28 individuals), 2 weeks (T2; n = 38 individuals) and 10 weeks (T3; n =33 individuals) after 2nd vaccination] and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages, stimulated with the SP S2 subunit SP are shown.


Figure 3B . Phospho - SYK Immunofluorescence - microscopy in macrophages before and after vaccination . Nuclei stained with DAPI . Scale bars indicate 20 µm . C . Quantification of pSYK positive macrophages by flow cytometry before ( n = 3 individuals ) and 14 d after vaccination ( n = 3 individuals ) . Total SYK was measured as control . D . Western blot analysis of phospho - NF - κB and NLRP3 in cell lysates of macrophages ( T0 ; T2 ) stimulated with LPS / N or SP / N . E . Macrophages were incubated with IKK - β - inhibitor KINK - 1 ( n = 10 individuals ) and SYK - inhibitors entospletinib ( n = 20 individuals ) , R406 ( n = 13 individuals ) prior to SP / nigericin stimulation . IL - 1β concentrations measured with ELISA . DMSO - treated , SP / N - stimulated and untreated unstimulated macrophages were used as control ( both n = 44 individuals ) . F . IL - 1β quantification in THP - 1 wild - type cells incubated with SYK inhibitors entospletinib ( n = 4 independent experiments ) and R406 ( n = 2 independent experiments ) or in THP - 1 SYKKO cells stimulated with SP / N ( n = 5 independent experiments ) . G . ASC formation quantified by immunofluorescence - microscopy in SP / N stimulated primary macrophages after vaccination ( n = 5 individuals ) . H . Quantification of cell death in T2 macrophages ( n = 4 individuals ) treated with R406 prior to SP / N - stimulation . I - K . mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages without inhibitor ( I [ exemplary results ] , J [ T0 : n = 5 individuals ; T2 : n = 5 individuals ] ) mROS quantified MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages with R406 treatment ( n = 4 individuals ) ( K ) . L . Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP / N or LPS / N . M . Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester ( TMRM ) stained macrophages at T0 ( n = 5 individuals ) and T2 ( n = 5 individuals ) stimulated with SP / N . N . T2 - macrophages ( n = 4 individuals ) were treated with the SYK - inhibitor R406 . Data information : For statistical analysis , one - or two - way ANOVA with tukey post hoc test was used . Box plots indicate the median and the upper and lower quartile . Outliers are plotted as individual dots ( outside the 10 - 90 percentile ) . Scatter dot plots show mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 . ( S - protein : SP , lipopolysaccharide : LPS , nigericin : N )

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Figure 3B. Phospho-SYK Immunofluorescence-microscopy in macrophages before and after vaccination. Nuclei stained with DAPI. Scale bars indicate 20 µm.C. Quantification of pSYK positive macrophages by flow cytometry before (n = 3 individuals) and 14 d after vaccination (n = 3 individuals). Total SYK was measured as control.D. Western blot analysis of phospho-NF-κB and NLRP3 in cell lysates of macrophages (T0; T2) stimulated with LPS/N or SP/N.E. Macrophages were incubated with IKK-β-inhibitor KINK-1 (n = 10 individuals) and SYK-inhibitors entospletinib (n = 20 individuals), R406 (n = 13 individuals) prior to SP/nigericin stimulation. IL-1β concentrations measured with ELISA. DMSO-treated, SP/N-stimulated and untreated unstimulated macrophages were used as control (both n = 44 individuals).F. IL-1β quantification in THP-1 wild-type cells incubated with SYK inhibitors entospletinib (n = 4 independent experiments) and R406 (n = 2 independent experiments) or in THP-1 SYKKO cells stimulated with SP/N (n = 5 independent experiments).G. ASC formation quantified by immunofluorescence-microscopy in SP/N stimulated primary macrophages after vaccination (n = 5 individuals).H. Quantification of cell death in T2 macrophages (n = 4 individuals) treated with R406 prior to SP/N-stimulation.I-K. mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages without inhibitor (I [exemplary results], J [T0: n = 5 individuals; T2: n = 5 individuals])mROS quantified MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages with R406 treatment (n = 4 individuals) (K).L. Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP/N or LPS/N.M. Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester (TMRM) stained macrophages at T0 (n = 5 individuals) and T2 (n = 5 individuals) stimulated with SP/N. N. T2-macrophages (n = 4 individuals) were treated with the SYK-inhibitor R406. Data information: For statistical analysis, one- or two-way ANOVA with tukey post hoc test was used. Box plots indicate the median and the upper and lower quartile. Outliers are plotted as individual dots (outside the 10-90 percentile). Scatter dot plots show mean ± SD. *p<0.05; **p<0.01, ***p<0.001. (S-protein: SP, lipopolysaccharide: LPS, nigericin: N)


Figure 4A - H . ( A ) T cells at T0 ( n = 6 individuals ) and T2 ( n = 10 individuals ) were incubated with SP / N - stimulated macrophages . Co - cultures of unstimulated macrophages were used as control ( con ) . CD4 + subpopulation analyses performed with flow cytometry ( Experimental setup : Fig EV3 ) . ( B ) Quantification of fold change of the CD4 + T cell subpopulation upon SP / N stimulation ( T0 ; T2 ) . Dotted line indicates the baseline ( fold change = 1 . 0 ) . ( C ) Fold change of CD4 + naïve T cell and CD4 + effector memory T cell populations ( D ) upon co - culture of macrophages stimulated with SP / N in presence of MCC950 or R406 . Identical analyses performed with CD8 + T cells ( E , F , G , H ) . I , J . PD - 1 expression was analyzed in CD4 + ( I ) and CD8 + T cells ( J ) by flow cytometry upon co - culture with SP / N - stimulated T0 ( n = 6 individuals ) or T2 ( n = 11 individuals ) macrophages . Co - cultures of unstimulated macrophages were used as control . K , L . Interferon - γ quantified in supernatants of corresponding co - cultures , untreated ( T0 [ n = 6 individuals ] and T2 [ n = 11 individuals ] ) ( K ) and treated with MCC950 or R406 ( L ) at T2 ( n = 11 individuals ) . M . IL - 1β concentrations determined in SP / N - stimulated monocyte - derived macrophages at 34 weeks after 2nd vaccination ( first booster ) ( T4 = 7 individuals ) and 2 weeks after the 3rd vaccination ( second booster ) ( T5 = 11 individuals ) .

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Figure 4A-H. (A) T cells at T0 (n = 6 individuals) and T2 (n = 10 individuals) were incubated with SP/N-stimulated macrophages. Co-cultures of unstimulated macrophages were used as control (con). CD4+ subpopulation analyses performed with flow cytometry (Experimental setup: Fig EV3). (B) Quantification of fold change of the CD4+ T cell subpopulation upon SP/N stimulation (T0; T2). Dotted line indicates the baseline (fold change = 1.0). (C) Fold change of CD4+ naïve T cell and CD4+ effector memory T cell populations (D) upon co-culture of macrophages stimulated with SP/N in presence of MCC950 or R406. Identical analyses performed with CD8+ T cells (E, F, G, H).I, J. PD-1 expression was analyzed in CD4+ (I) and CD8+ T cells (J) by flow cytometry upon co-culture with SP/N-stimulated T0 (n = 6 individuals) or T2 (n = 11 individuals) macrophages. Co-cultures of unstimulated macrophages were used as control.K, L. Interferon-γ quantified in supernatants of corresponding co-cultures, untreated (T0 [n = 6 individuals] and T2 [n = 11 individuals]) (K) and treated with MCC950 or R406 (L) at T2 (n = 11 individuals).M. IL-1β concentrations determined in SP/N-stimulated monocyte-derived macrophages at 34 weeks after 2nd vaccination (first booster) (T4 = 7 individuals) and 2 weeks after the 3rd vaccination (second booster) (T5 = 11 individuals).


Figure 2 ( A ) Recombinant proteins were mixed , as indicated , in the presence ( lane 5 ) or absence ( lane 4 ) of ATP and incubated for 1 h at 30 ° C before being separated by SDS - PAGE and analyzed by western blotting using the α - His antibody . Components present ( + ) or absent ( − ) from the assay are indicated . ( B ) Recombinant proteins were mixed , as indicated , and incubated as above . The reactions were stopped , subjected to SDS - PAGE and stained with Coomassie Blue . ( C ) Recombinant proteins were mixed , as indicated , incubated as above and analyzed with α - His antibody .

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Figure 2(A) Recombinant proteins were mixed, as indicated, in the presence (lane 5) or absence (lane 4) of ATP and incubated for 1 h at 30°C before being separated by SDS-PAGE and analyzed by western blotting using the α-His antibody. Components present (+) or absent (−) from the assay are indicated.(B) Recombinant proteins were mixed, as indicated, and incubated as above. The reactions were stopped, subjected to SDS-PAGE and stained with Coomassie Blue.(C) Recombinant proteins were mixed, as indicated, incubated as above and analyzed with α-His antibody.


Figure 3 ( A ) The occurrence of mC - ATG5 puncta in WT L . major promastigotes expressing mC - ATG5 incubated in nutrient - rich medium at log phase at 26 ° C . ( B ) The multiplicity of puncta in promastigotes expressing mC - ATG5 after starvation for up to 1 h in PBS at 26 ° C . The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken . ( C ) Co - labelling of puncta with mC - ATG5 and GFP - ATG12 co - expressed in promastigotes incubated in PBS for 2 h at 26 ° C . ( D ) In nutrient - rich medium , GFP - ATG8 punctum without mC - ATG5 staining is arrowed . ( E ) Incubated in PBS for 30 min at 26 ° C . ( F ) In nutrient - rich medium at 26 ° C . ( G ) Incubated in PBS for 30 min at 26 ° C , the small panels are enlargements of the merged panels . ( H - I ) Promastigotes in nutrient - rich medium . ( J - K ) Promastigotes at late log phase in nutrient - rich conditions . ( L ) Promastigotes at stationary phase in nutrient - rich conditions . Scale bar throughout , 10 µm .

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Figure 3(A) The occurrence of mC-ATG5 puncta in WT L. major promastigotes expressing mC-ATG5 incubated in nutrient-rich medium at log phase at 26°C.(B) The multiplicity of puncta in promastigotes expressing mC-ATG5 after starvation for up to 1 h in PBS at 26°C. The time ranges indicated reflect the starvation period together with the 30 min period during which the observations on the microscope were undertaken.(C) Co-labelling of puncta with mC-ATG5 and GFP-ATG12 co-expressed in promastigotes incubated in PBS for 2 h at 26°C. (D) In nutrient-rich medium, GFP-ATG8 punctum without mC-ATG5 staining is arrowed. (E) Incubated in PBS for 30 min at 26°C. (F) In nutrient-rich medium at 26°C. (G) Incubated in PBS for 30 min at 26°C, the small panels are enlargements of the merged panels. (H-I) Promastigotes in nutrient-rich medium. (J-K) Promastigotes at late log phase in nutrient-rich conditions. (L) Promastigotes at stationary phase in nutrient-rich conditions. Scale bar throughout, 10 µm.


Figure 4 ( A ) Growth curve of Leishmania promastigotes in HOMEM medium at 26 ° C . * , Δatg5 differed significantly from WT ( p < 0 . 05 ) . ( B ) The occurrence of GFP - ATG8 puncta in promastigotes after incubation in nutrient - deprived ( PBS , ND ) and nutrient - rich ( HOMEM medium , NR ) conditions for 2 h at 26 ° C . Scale bar , 10 µm ( C ) Occurrence of GFP - ATG8 puncta in promastigotes when incubated in the conditions detailed in ( B ) . Means ± SD from four independent experiments . * and * * , occurrence of GFP - ATG8 puncta in Δatg5 were significantly different from in WT in nutrient - deprived and nutrient - rich conditions ( p < 0 . 05 ) . ( D ) Western blot analysis of extracts of promastigotes expressing GFP - ATG8 at logarithmic growth under standard conditions and probed with α - GFP antibody . The faster migrating , lipidated band is labelled GFP - ATG8 - II while the un - lipidated band migrating more slowly is labelled GFP - ATG8 - I .

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Figure 4(A) Growth curve of Leishmania promastigotes in HOMEM medium at 26°C. *, Δatg5 differed significantly from WT (p<0.05).(B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm(C) Occurrence of GFP-ATG8 puncta in promastigotes when incubated in the conditions detailed in (B). Means ± SD from four independent experiments. * and **, occurrence of GFP-ATG8 puncta in Δatg5 were significantly different from in WT in nutrient-deprived and nutrient-rich conditions (p<0.05).(D) Western blot analysis of extracts of promastigotes expressing GFP-ATG8 at logarithmic growth under standard conditions and probed with α-GFP antibody. The faster migrating, lipidated band is labelled GFP-ATG8-II while the un-lipidated band migrating more slowly is labelled GFP-ATG8-I.


Figure 5 ( A ) Enlarged ( EM ) and swollen ( WM ) mitochondria seen by transmission electron microscopy ( TEM ) in Δatg5 promastigotes under standard growth conditions . WT is shown at bottom right . Scale bar , 500 nm . ( B ) Fluorescent intensity from MitoTracker Red ( MTR , 0 . 1 µM ) and MitoTracker Green ( MTG , 0 . 2 µM ) in 2 × 106promastigotes after 30 min incubation at 26 ° C . Values shown are the means ± SD from three independent experiments . * and * * , fluorescence was significantly different between WT and Δatg5 ( p < 0 . 05 ) . ( C ) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP - GFP . Scale bar , 10 µm . ( D ) Differential staining of promastigotes with both MTR ( 0 . 1 µM ) and MTG ( 0 . 2 µM ) . Scale bar , 10 µm . ( E ) Viability , as measured by Alamar Blue reduction , of promastigotes . All data are means ± SD from three independent experiments . * , Alamar blue reduction was significantly different ( p < 0 . 05 ) . ( F ) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2 × 106 / ml with H2DCFDA at 0 . 1 mM for 2 h at 26 ° C . Values shown are the means ± SD from three independent experiments . * , DCF fluorescence was significantly different ( p < 0 . 05 ) .

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Figure 5(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm.(B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05).(C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm.(D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm.(E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05).(F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).


Figure 6Phospholipid accumulation in Δatg5 promastigotes . Negative ion survey scans ( 650 - 900 m / z ) of WT ( A ) and Δatg5 ( B ) promastigotes extracted for lipids and analysed by ES - MS , as described in Materials and Methods . a = ( alkylacyl ) .

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Figure 6Phospholipid accumulation in Δatg5 promastigotes.Negative ion survey scans (650-900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).


Figure 7 ( A ) Proportion of metacyclic promastigotes in stationary phase cultures , assessed using the PNA assay . Values shown are the means ± SD from three independent experiments . * , differed significantly ( p < 0 . 05 ) . ( B ) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α - HASPB . α - Cysteine Synthase was used as a loading control [ 56 ] . ( C ) Infectivity and survival of promastigotes in peritoneal macrophages in vitro , infected at a ratio 5 ∶ 1 , with the infection rates being assessed after 1 and 5 days . * , differed significantly ( p < 0 . 01 ) . ( D ) Lesion progression in BALB / c mice inoculated with 5 × 105 stationary phase promastigotes . Values shown are the means ± SD from 5 mice . * , infection level of Δatg5 differed significantly from WT ( p < 0 . 01 ) and Δatg5 : : ATG5 ( p < 0 . 05 ) . ( E ) Morphologies of cells isolated from mouse lesions and analysed by SEM . Scale bar : 2 µm .

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Figure 7(A) Proportion of metacyclic promastigotes in stationary phase cultures, assessed using the PNA assay. Values shown are the means ± SD from three independent experiments. *, differed significantly (p<0.05).(B) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α-HASPB. α-Cysteine Synthase was used as a loading control [56].(C) Infectivity and survival of promastigotes in peritoneal macrophages in vitro, infected at a ratio 5∶1, with the infection rates being assessed after 1 and 5 days. *, differed significantly (p<0.01).(D) Lesion progression in BALB/c mice inoculated with 5×105 stationary phase promastigotes. Values shown are the means ± SD from 5 mice. *, infection level of Δatg5 differed significantly from WT (p<0.01) and Δatg5::ATG5 (p<0.05).(E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.


Figure 8A ) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient - rich medium . Shown are ovoid and amastigote - like form ( left ) ; spindled - shaped form without an external flagellum ( centre left panel ) and with an external flagellum of varying lengths ( centre right and right panels ) . Scale bar , 10 µm . ( B ) Distribution of flagella lengths and body lengths of stationary phase promastigotes . Data represent measurements from ∼ 200 cells from each promastigote population .

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Figure 8A) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient-rich medium. Shown are ovoid and amastigote-like form (left); spindled-shaped form without an external flagellum (centre left panel) and with an external flagellum of varying lengths (centre right and right panels). Scale bar, 10 µm.(B) Distribution of flagella lengths and body lengths of stationary phase promastigotes. Data represent measurements from ∼200 cells from each promastigote population.


Figure 1B ) Tumour growth curves for successive four - generation xenografts treated with 5 - Fu or PBS . Twelve mice per group . C ) Representative cases show the first and fourth generation xenograft H & E staining ( left ) and EBER - 1 ISH staining ( right ) . D ) The morphology of parental SNU719 and SNU - 4th cells . E ) Tumour sphere culture was performed to evaluate the sphere formation capacity in SNU719 and SNU - 4th cells , and the data are shown as the mean numbers and sizes of the tumour spheres . The central horizontal lines represent the median , the top and the bottom positions of the box represent upper and lower quartiles , error bars represent the mean ± SD , and the average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . F ) CCK8 assay of SNU719 and SNU - 4th cells to determine cell proliferation under the indicated culture conditions ( 10 % FBS or 0 . 1 % FBS ) . Expression of stemness markers ( Sox2 , Klf4 , Bmi1 , Oct4 ) and drug resistance genes ( ABCG2 , Mrp1 ) evaluated by real - time PCR ( G ) in SNU719 and SNU - 4th cells . Expression of stemness markers ( Sox2 , Klf4 , Bmi1 , Oct4 ) and drug resistance genes ( ABCG2 , Mrp1 ) evaluated by WB ( H ) in SNU719 and SNU - 4th cells . I ) Real - time PCR analysis of the expression of EMT - associated markers ( E - cad , vimentin ) and transcription factors ( Twist1 , Snail , Slug , Zeb1 ) in SNU719 and SNU - 4th cells . J ) WB analysis of the expression of E - cad , vimentin and Mmp7 in SNU719 and SNU - 4th cells . K ) Colony formation assay analysis of the colony - forming ability in SNU719 and SNU - 4th cells . L ) Transwell migration and the Matrigel invasion assay were performed to assess the migratory and invasive capability of SNU719 and SNU - 4th cells . M ) Representative images of the flow cytometric analysis of SP cells . N ) Representative images of the flow cytometric analysis of the surface antigen phenotype .

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Figure 1B) Tumour growth curves for successive four-generation xenografts treated with 5-Fu or PBS. Twelve mice per group.C) Representative cases show the first and fourth generation xenograft H&E staining (left) and EBER-1 ISH staining (right).D) The morphology of parental SNU719 and SNU-4th cells.E) Tumour sphere culture was performed to evaluate the sphere formation capacity in SNU719 and SNU-4th cells, and the data are shown as the mean numbers and sizes of the tumour spheres. The central horizontal lines represent the median, the top and the bottom positions of the box represent upper and lower quartiles, error bars represent the mean ± SD, and the average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates.F) CCK8 assay of SNU719 and SNU-4th cells to determine cell proliferation under the indicated culture conditions (10% FBS or 0.1% FBS).Expression of stemness markers (Sox2, Klf4, Bmi1, Oct4) and drug resistance genes (ABCG2, Mrp1) evaluated by real-time PCR (G) in SNU719 and SNU-4th cells.Expression of stemness markers (Sox2, Klf4, Bmi1, Oct4) and drug resistance genes (ABCG2, Mrp1) evaluated by WB (H) in SNU719 and SNU-4th cells.I) Real-time PCR analysis of the expression of EMT-associated markers (E-cad, vimentin) and transcription factors (Twist1, Snail, Slug, Zeb1) in SNU719 and SNU-4th cells.J) WB analysis of the expression of E-cad, vimentin and Mmp7 in SNU719 and SNU-4th cells.K) Colony formation assay analysis of the colony-forming ability in SNU719 and SNU-4th cells.L) Transwell migration and the Matrigel invasion assay were performed to assess the migratory and invasive capability of SNU719 and SNU-4th cells.M) Representative images of the flow cytometric analysis of SP cells.N) Representative images of the flow cytometric analysis of the surface antigen phenotype.


Figure 2A ) Heat map analysis of the expression of circRNAs ( both human circRNAs and ebv - circRNAs ) in the fourth passage xenografts treated with 5 - Fu or PBS . Red colour represents upregulated circRNAs , and green colour represents downregulated circRNAs . ( B ) Volcano plot representing the expression fold changes in circRNAs . The red dots represent upregulated circRNAs , and green dots represent downregulated circRNAs . ( C ) The back - spliced read distribution of ebv - circRNAs ( n = 261 ) . ( D ) The length distribution of ebv - circRNAs . ( E ) Genomic origin of ebv - circRNAs . ( F ) Number of ebv - circRNAs produced from one gene .

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Figure 2A) Heat map analysis of the expression of circRNAs (both human circRNAs and ebv-circRNAs) in the fourth passage xenografts treated with 5-Fu or PBS. Red colour represents upregulated circRNAs, and green colour represents downregulated circRNAs.(B) Volcano plot representing the expression fold changes in circRNAs. The red dots represent upregulated circRNAs, and green dots represent downregulated circRNAs. (C) The back-spliced read distribution of ebv-circRNAs (n=261). (D) The length distribution of ebv-circRNAs.(E) Genomic origin of ebv-circRNAs.(F) Number of ebv-circRNAs produced from one gene.


Figure 3 ( B ) Real - time PCR analysis of the expression of circLMP2A in SNU - 4th , SNU719 and YCCEL1 cells . ( C ) The existence of circLMP2A was validated by RT - PCR ( up ) and real - time PCR ( down ) in SNU - 4th , SNU719 and YCCEL1 cells treated with or without RNase R digestion . GAPDH was used as a negative control . ( D ) Real - time PCR analysis of circLMP2A and LMP2A expression after treatment with actinomycin D at the indicated time points in SNU - 4th cells . ( E ) Real - time PCR analysis of circLMP2A and LMP2A expression in either the cytoplasm or nucleus in SNU - 4th cells . GAPDH and U6 were used as controls in cytoplasm and nucleus , respectively . ( F ) RNA ISH for circLMP2A . U6 and 18S are mainly localized in the nucleus and cytoplasm , respectively , and were used as controls . ( G ) Kaplan - Meier survival curve analysis showing the correlation between circLMP2A expression and OS . n = 69 , P < 0 . 001 , log - rank test . ( H ) Kaplan - Meier survival curve analysis showing the correlation between circLMP2A expression and progression - free survival ( PFS ) . n = 69 , P < 0 . 001 , log - rank test .

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Figure 3(B) Real-time PCR analysis of the expression of circLMP2A in SNU-4th, SNU719 and YCCEL1 cells.(C) The existence of circLMP2A was validated by RT-PCR (up) and real-time PCR (down) in SNU-4th, SNU719 and YCCEL1 cells treated with or without RNase R digestion. GAPDH was used as a negative control.(D) Real-time PCR analysis of circLMP2A and LMP2A expression after treatment with actinomycin D at the indicated time points in SNU-4th cells.(E) Real-time PCR analysis of circLMP2A and LMP2A expression in either the cytoplasm or nucleus in SNU-4th cells. GAPDH and U6 were used as controls in cytoplasm and nucleus, respectively.(F) RNA ISH for circLMP2A. U6 and 18S are mainly localized in the nucleus and cytoplasm, respectively, and were used as controls.(G) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and OS. n= 69, P < 0.001, log-rank test. (H) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and progression-free survival (PFS). n = 69, P < 0.001, log-rank test.


Figure 4 ( A ) Real - time PCR analysis of the interfering efficacy and linear LMP2A mRNA levels after transfection of three circLMP2A shRNAs . ( B ) Tumour sphere culture analysis of the tumour sphere formation in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . The central horizontal lines represent the median , top and bottom positions of the box represent upper and lower quartiles , and error bars represent the mean ± SD . The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . Scale bars = 100µm . EMT - related markers , stemness markers and drug resistance genes were evaluated by real - time PCR ( C ) in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . EMT - related markers , stemness markers and drug resistance genes were evaluated by WB ( D ) in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . ( E - F ) Transwell assay ( E ) and colony formation assay ( F ) analysis of the migratory and invasive capability and colony - forming ability in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . Scale bars = 100µm . ( G - H ) Flow cytometric analysis of the rate of SP cells ( G ) and apoptosis ( H ) in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . Subcutaneously established tumour xenografts in NOD / SCID mice . Tumour volume ( I ) of the xenografts were remarkably inhibited by down - regulation of circLMP2A . Six mice per group . Subcutaneously established tumour xenografts in NOD / SCID mice . tumour weight ( J ) of the xenografts were remarkably inhibited by down - regulation of circLMP2A . Six mice per group . ( K - L ) Representative images of bioluminescence ( K ) and lung metastases ( L ) are presented to measure the metastatic colonies . Six mice per group , scale bars = 50µm .

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Figure 4(A) Real-time PCR analysis of the interfering efficacy and linear LMP2A mRNA levels after transfection of three circLMP2A shRNAs.(B) Tumour sphere culture analysis of the tumour sphere formation in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. The central horizontal lines represent the median, top and bottom positions of the box represent upper and lower quartiles, and error bars represent the mean ± SD. The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates. Scale bars =100µm.EMT-related markers, stemness markers and drug resistance genes were evaluated by real-time PCR (C) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.EMT-related markers, stemness markers and drug resistance genes were evaluated by WB (D) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.(E-F) Transwell assay (E) and colony formation assay (F) analysis of the migratory and invasive capability and colony-forming ability in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. Scale bars =100µm.(G-H) Flow cytometric analysis of the rate of SP cells (G) and apoptosis (H) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.Subcutaneously established tumour xenografts in NOD/SCID mice. Tumour volume (I) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group.Subcutaneously established tumour xenografts in NOD/SCID mice. tumour weight (J) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group.(K-L) Representative images of bioluminescence (K) and lung metastases (L) are presented to measure the metastatic colonies. Six mice per group, scale bars =50µm.


Figure 5 ( A ) Real - time PCR confirmed the over - expression of circLMP2A in SNU719 and YCCEL1 cells after transfection . ( B ) Over - expression of circLMP2A induced the formation of tumour spheres in SNU719 and YCCEL1 cells . The central horizontal lines represent the median , top and bottom positions of the box represent upper and lower quartiles , and error bars represent the mean ± SD . The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . Scale bars = 100µm . EMT - related markers , stemness markers and drug resistance genes were assessed by real - time PCR ( C ) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . EMT - related markers , stemness markers and drug resistance genes were assessed by WB ( D ) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . ( E - F ) Flow cytometric analysis of the rate of SP cells ( E ) and apoptosis ( F ) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . ( G - H ) Transwell assay ( G ) and colony formation assay ( H ) analysis of the migratory and invasive capability and colony - forming ability in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . Scale bars = 100µm . Subcutaneously established tumour xenografts in NOD / SCID mice . Tumour volume ( I ) of the xenografts were significantly increased by over - expression of circLMP2A . Six mice per group . Subcutaneously established tumour xenografts in NOD / SCID mice . tumour weight ( J ) of the xenografts were significantly increased by over - expression of circLMP2A . Six mice per group . ( K - L ) Representative images of bioluminescence ( K ) and lung metastases ( L ) are presented to measure the metastatic colonies . Six mice per group , scale bars = 50µm .

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Figure 5(A) Real-time PCR confirmed the over-expression of circLMP2A in SNU719 and YCCEL1 cells after transfection.(B) Over-expression of circLMP2A induced the formation of tumour spheres in SNU719 and YCCEL1 cells. The central horizontal lines represent the median, top and bottom positions of the box represent upper and lower quartiles, and error bars represent the mean ± SD. The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates. Scale bars =100µm.EMT-related markers, stemness markers and drug resistance genes were assessed by real-time PCR (C) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.EMT-related markers, stemness markers and drug resistance genes were assessed by WB (D) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.(E-F) Flow cytometric analysis of the rate of SP cells (E) and apoptosis (F) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.(G-H) Transwell assay (G) and colony formation assay (H) analysis of the migratory and invasive capability and colony-forming ability in SNU719 and YCCEL1 cells transfected with circLMP2A or vector. Scale bars =100µm.Subcutaneously established tumour xenografts in NOD/SCID mice. Tumour volume (I) of the xenografts were significantly increased by over-expression of circLMP2A. Six mice per group.Subcutaneously established tumour xenografts in NOD/SCID mice. tumour weight (J) of the xenografts were significantly increased by over-expression of circLMP2A. Six mice per group.(K-L) Representative images of bioluminescence (K) and lung metastases (L) are presented to measure the metastatic colonies. Six mice per group, scale bars =50µm.


Figure 6 ( B ) The relative levels of 7 miRNA candidates were detected by real - time PCR in SNU719 and SNU - 4th cells . ( C - E ) Real - time PCR analysis of 7 miRNA expression in SNU - 4th cells transfected with sh - circLMP2A - 2 or sh - control ( C ) , and SNU719 ( D ) and YCCEL1 cells ( E ) transfected with circLMP2A or vector . ( F - H ) circ LMP2Ain specific probe and detected by real - time PCR ( F ) and RT - PCR ( H ) . The relative levels of 5 miRNA candidates were detected by real - time PCR ( G ) and RT - PCR ( H ) . GAPDH was used as a negative control . ( I ) RNA ISH showing the co - localization between circLMP2A and miR - 3908 in SNU - 4th cells . circLMP2A probes were labelled with Cy3 , and miR - 3908 probes were labelled with FITC . Nuclei were stained with DAPI . ( K ) Dual luciferase reporter assay for luciferase activity of the indicated plasmids ( circLMP2A Wt , circLMP2A mut1 , circLMP2A mut2 , circLMP2Amut3 luciferase reporter ) in HEK - 293T cells transfected with mimic - NC or miR - 3908 .

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Figure 6(B) The relative levels of 7 miRNA candidates were detected by real-time PCR in SNU719 and SNU-4th cells.(C-E) Real-time PCR analysis of 7 miRNA expression in SNU-4th cells transfected with sh-circLMP2A-2 or sh-control (C), and SNU719 (D) and YCCEL1 cells (E) transfected with circLMP2A or vector.(F-H) circ LMP2Ain specific probe and detected by real-time PCR (F) and RT-PCR (H). The relative levels of 5 miRNA candidates were detected by real-time PCR (G) and RT-PCR (H). GAPDH was used as a negative control.(I) RNA ISH showing the co-localization between circLMP2A and miR-3908 in SNU-4th cells. circLMP2A probes were labelled with Cy3, and miR-3908 probes were labelled with FITC. Nuclei were stained with DAPI.(K) Dual luciferase reporter assay for luciferase activity of the indicated plasmids (circLMP2A Wt, circLMP2A mut1, circLMP2A mut2, circLMP2Amut3 luciferase reporter) in HEK-293T cells transfected with mimic-NC or miR-3908.