Figure 2 ( A , B ) Co - localization analysis of neosynthesized alcAp - UapA - GFP with early ( SedV ) or late ( PHosbp ) markers , respectively , tagged with mCherry or mRFP . Images show single hyphal cells . For conditions of transcriptional derepression see Materials and methods . Quantification of co - localization was performed by calculating Pearson ' s Correlation Coefficient ( PCC ) . One sample t - test was performed to test the significance of differences in PCCs . Biological / technical replicates : 2 / 9 for alcAp - UapA - GFP mCherry - SedV and 2 / 8 for alcAp - UapA - GFP mRFP - PHosbp . For the definition of the two categories of replicates see Materials and methods . Results of quantification , shown on the middle , suggest that there is no significant overlapping fluorescent signal of UapA with SedV ( PCC = 0 . 25 ± 0 . 09 , P < 0 . 0001 ) or with PHOSBP ( PCC = 0 . 34 ± 0 . 06 , P < 0 . 0001 ) , as PCC values close to 0 . 2 - 0 . 3 are also commonly obtained when distinct compartments ( e . g . ER and early Golgi or early and late Golgi ) are followed with different fluorophores . Scale bars : 2 μm . ( C , D ) Co - localization analysis of neosynthesized alcAp - GFP - SynA , used as a conventional cargo that traffics through the Golgi , with early ( SedV ) or late ( PHospb ) markers tagged with mCherry or mRFP , respectively . Images show single hyphal cells . For conditions of transcriptional derepression see Materials and methods . Quantification of co - localization and statistical analysis was performed as in ( A , B ) . Biological / technical replicates : 2 / 10 for each strain . Statistical analysis showed significant colocalization of SynA with the late Golgi marker ( PCC = 0 . 67 ± 0 . 04 , P < 0 . 0001 ) , but not with early Golgi marker ( PCC = 0 . 37 ± 0 . 06 , P < 0 . 0001 ) . An explanation for lack of co - localization of SynA with SedV is given in the text . Scale bars : 2 μm . The cartoon at the bottom depicts the localization of early or late markers as established here in several previous

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 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

Figure 2(A, B) Co-localization analysis of neosynthesized alcAp-UapA-GFP with early (SedV) or late (PHosbp) markers, respectively, tagged with mCherry or mRFP. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization was performed by calculating Pearson's Correlation Coefficient (PCC). One sample t-test was performed to test the significance of differences in PCCs. Biological/technical replicates:2/9 for alcAp-UapA-GFP mCherry-SedV and 2/8 for alcAp-UapA-GFP mRFP-PHosbp. For the definition of the two categories of replicates see Materials and methods. Results of quantification, shown on the middle, suggest that there is no significant overlapping fluorescent signal of UapA with SedV (PCC=0.25±0.09, P<0.0001) or with PHOSBP (PCC=0.34±0.06, P<0.0001), as PCC values close to 0.2-0.3 are also commonly obtained when distinct compartments (e.g. ER and early Golgi or early and late Golgi) are followed with different fluorophores. Scale bars: 2 μm.(C, D) Co-localization analysis of neosynthesized alcAp-GFP-SynA, used as a conventional cargo that traffics through the Golgi, with early (SedV) or late (PHospb) markers tagged with mCherry or mRFP, respectively. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization and statistical analysis was performed as in (A, B). Biological/technical replicates: 2/10 for each strain. Statistical analysis showed significant colocalization of SynA with the late Golgi marker (PCC = 0.67±0.04, P<0.0001), but not with early Golgi marker (PCC=0.37±0.06, P<0.0001). An explanation for lack of co-localization of SynA with SedV is given in the text. Scale bars: 2 μm. The cartoon at the bottom depicts the localization of early or late markers as established here in several previous studies


Figure 3 ( B ) Key endogenous genes controlling Golgi ( sedV , geaA , hypB ) or post - Golgi trafficking ( rabE , ap1σ , claH ) were genetically replaced by versions transcribed under the highly repressible thiAp promoter via targeted homologous recombination . In the absence of thiamine from the growth medium ( derepressed conditions ) the relative proteins are expressed , while upon addition of thiamine at the onset of conidiospore germination ( ab initio repression ) the expression of these proteins is tightly repressed . Proteins are detected by a standard western blot analysis using either anti - FLAG or anti - GFP antibodies for Golgi and Post - Golgi proteins . Equal loading and protein steady state levels are normalized against the amount of actin , detected with a specific antibody . ( C ) In the absence of thiamine from the growth medium ( derepressed conditions ) the corresponding strains grow nearly as an isogenic wild - type control , although a delay in growth is observed for thiAp - sedV and less so for thiAp - ap1σ ( upper row ) . In the presence of thiamine to the growth medium most cells do not form colonies , except for thiAp - hypB which forms a compact slow growing colony ( lower row ) . ( D ) Microscopic examination of the corresponding strains under thiamine ( repressing conditions ) shows that , in most cases , the apical region of germlings is enlarged and growth is arrested . This morphological phenotype , taken as a strong indication of blocked secretion , is more evident in thiAp - sedV and thiAp - rabE but concerns all strains , except for thiAp - hypB . Scale bar : 5 μm . ( E ) Subcellular localization of UapA , after 6 - 8 hours of initiation of transcription , via its native uapA promoter , while conventional sedV , geaA or hypB transcription is repressed by thiamine ab initio . Scale bars : 2 μm . Notice that UapA - GFP translocates in the PM of germlings in all cases . Results shown are confirmed by quantification ( right panel ) of UapA - GFP PM / cytosolic intensity ratios for the four strains ( for details see Materials and methods ) . Mean PM / cytosolic intensity ratios for wild - type , thiAp - sedV , thiAp - geaA and thiAp - hypB are 0 . 53 ± 0 . 02 , 0 . 52 ± 0 . 02 , 0 . 52 ± 0 . 02 and 0 . 52 ± 0 . 02 , respectively . For the statistical analysis , Tukey ' s Multiple Comparison test was performed ( One - way ANOVA ) . No statistical significance was found between the wild - type and each of the mutant strains . Biological / technical replicates : 3 / 15 for wild - type , 3 / 18 for thiAp - sedV , 3 / 15 for thiAp - geaA and 3 / 18 for thiAp - hypB . ( F ) alcAp - UapA - GFP , under derepressing conditions , translocates to the PM of a strain carrying a thermosensitive mutation in SedVts both at the permissive ( 25oC ) and restrictive temperature ( 42oC ) . A degree of increased degradation of UapA , caused by exposure to high temperature ( 42oC ) , is apparent as cytosolic puncta , which correspond to membrane aggregates and sorting to vacuoles . Scale bar : 5 μm . ( G ) Unlike UapA , de novo made apical markers , such as ChsB or SynA , lose their polar localization , when the expression of sedV , geaA or hypB is repressed . Scale bars : 2 μm . Quantification : GFP - ChsB PM / cytosolic and alcAp - GFP - SynA PM / cytosolic intensity ratios are plotted to the right upper and lower panel , respectively . For GFP - ChsB , mean PM / cytosolic intensity ratios are : 0 . 60 ± 0 . 02 ( wild - type ) , 0 . 11 ± 0 . 01 ( thiAp - sedV ) , 0 . 26 ± 0 . 02 ( thiAp - null ) and 0 . 34 ± 0 . 01 ( thiAp - hypB ) . For alcAp - GFP - SynA mean PM / cytosolic intensity ratios for wild - type , thiAp - sedV , thiAp - geaA and thiAp - hypB are 0 . 61 ± 0 . 03 , 0 . 14 ± 0 . 02 , 0 . 26 ± 0 . 03 and 0 . 39 ± 0 . 03 , respectively . The statistical analysis was performed as in ( E ) . A significant difference ( * * * * P < 0 . 0001 ) of GFP - ChsB fluorescence in the apical PM membrane was found between the wild - type and the three strains lacking the key - Golgi proteins . This is also the case for SynA , as seen in the scatter plot on the lower right panel , where the fluorescence intensity of SynA is diminished ( * * * * P < 0 . 0001 ) when sedV , geaA and hypB are repressed . Biological / technical replicates : 3 / 15 for each strain .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(B) Key endogenous genes controlling Golgi (sedV, geaA, hypB) or post-Golgi trafficking (rabE, ap1σ, claH) were genetically replaced by versions transcribed under the highly repressible thiAp promoter via targeted homologous recombination. In the absence of thiamine from the growth medium (derepressed conditions) the relative proteins are expressed, while upon addition of thiamine at the onset of conidiospore germination (ab initio repression) the expression of these proteins is tightly repressed. Proteins are detected by a standard western blot analysis using either anti-FLAG or anti-GFP antibodies for Golgi and Post-Golgi proteins. Equal loading and protein steady state levels are normalized against the amount of actin, detected with a specific antibody.(C) In the absence of thiamine from the growth medium (derepressed conditions) the corresponding strains grow nearly as an isogenic wild-type control, although a delay in growth is observed for thiAp-sedV and less so for thiAp-ap1σ (upper row). In the presence of thiamine to the growth medium most cells do not form colonies, except for thiAp-hypB which forms a compact slow growing colony (lower row).(D) Microscopic examination of the corresponding strains under thiamine (repressing conditions) shows that, in most cases, the apical region of germlings is enlarged and growth is arrested. This morphological phenotype, taken as a strong indication of blocked secretion, is more evident in thiAp-sedV and thiAp-rabE but concerns all strains, except for thiAp-hypB. Scale bar: 5 μm.(E) Subcellular localization of UapA, after 6-8 hours of initiation of transcription, via its native uapA promoter, while conventional sedV, geaA or hypB transcription is repressed by thiamine ab initio. Scale bars: 2 μm. Notice that UapA-GFP translocates in the PM of germlings in all cases. Results shown are confirmed by quantification (right panel) of UapA-GFP PM/cytosolic intensity ratios for the four strains (for details see Materials and methods). Mean PM/cytosolic intensity ratios for wild-type, thiAp-sedV, thiAp-geaA and thiAp-hypB are 0.53±0.02, 0.52±0.02, 0.52±0.02 and 0.52±0.02, respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). No statistical significance was found between the wild-type and each of the mutant strains. Biological/technical replicates: 3/15 for wild-type, 3/18 for thiAp-sedV, 3/15 for thiAp-geaA and 3/18 for thiAp-hypB.(F) alcAp-UapA-GFP, under derepressing conditions, translocates to the PM of a strain carrying a thermosensitive mutation in SedVts both at the permissive (25oC) and restrictive temperature (42oC). A degree of increased degradation of UapA, caused by exposure to high temperature (42oC), is apparent as cytosolic puncta, which correspond to membrane aggregates and sorting to vacuoles. Scale bar: 5 μm.(G) Unlike UapA, de novo made apical markers, such as ChsB or SynA, lose their polar localization, when the expression of sedV, geaA or hypB is repressed. Scale bars: 2 μm. Quantification: GFP-ChsB PM/cytosolic and alcAp-GFP-SynA PM/cytosolic intensity ratios are plotted to the right upper and lower panel, respectively. For GFP-ChsB, mean PM/cytosolic intensity ratios are: 0.60±0.02 (wild-type), 0.11±0.01 (thiAp-sedV), 0.26±0.02 (thiAp-null) and 0.34±0.01 (thiAp-hypB). For alcAp-GFP-SynA mean PM/cytosolic intensity ratios for wild-type, thiAp-sedV, thiAp-geaA and thiAp-hypB are 0.61±0.03, 0.14±0.02, 0.26±0.03 and 0.39±0.03, respectively. The statistical analysis was performed as in (E). A significant difference (****P<0.0001) of GFP-ChsB fluorescence in the apical PM membrane was found between the wild-type and the three strains lacking the key-Golgi proteins. This is also the case for SynA, as seen in the scatter plot on the lower right panel, where the fluorescence intensity of SynA is diminished (****P<0.0001) when sedV, geaA and hypB are repressed. Biological/technical replicates: 3/15 for each strain.


Figure 4 ( A ) Growth test showing that Sec24 expression is essential for growth as its transcriptional repression by thiamine ( + thi ) leads to absence of colony formation , despite initial germination The strains shown are isogenic except for the sec24 locus . thiAp - sec24 signifies the strain where the endogenous sec24 promoter was replaced by the thiAp promoter . ( B ) Epifluorescence microscopy analysis of the subcellular localization of UapA - GFP under conditions where sec24 transcription is ab initio derepressed ( upper panel ) or repressed by thiamine ( lower panel ) . o / n ( overnight ) means addition of thiamine from the onset of germination . Germination of conidiospores takes place until full repression of Sec24 is achieved ( 10 - 12 h ) . Notice the total lack of PM - associated signal of UapA under conditions of Sec24 repression . Scale bar : 5 μm . Quantification : In the scatter plot on the right , UapA - GFP PM / cytosolic intensity ratios are quantified when sec24 is derepressed ( - thi ) or repressed ( + thi ) . Mean PM / cytosolic intensity ratios are 0 . 55 ± 0 . 02 and 0 . 32 ± 0 . 02 respectively . To test the significance of differences , an unpaired t - test was performed , which verified the significant difference ( * * * * P < 0 . 0001 ) in the presence of thiamine . Biological / technical replicates : 2 / 15 for each condition . ( C ) Epifluorescence microscopy analysis of the subcellular localization of the apical marker chitin synthase ( GFP - ChsB ) under sec24 derepressed or repressed conditions . Notice the loss of apical depositioning and the concurrent labeling of cytoplasmic foci and membranous structures under repression conditions . Scale bar : 5 μm . Quantification : GFP - ChsB PM / cytosolic intensity ratios are plotted in cases where sec24 is derepressed ( - thi ) or repressed ( + thi ) . Mean values are 0 . 56 ± 0 . 03 and 0 . 12 ± 0 . 02 respectively . The fluorescence intensity of GFP - ChsB is statistically lower ( * * * * P < 0 . 0001 ) when the expression of sec24 is repressed ( statistical analysis as in B ) . Biological / technical replicates : 2 / 15 for each condition . ( D ) Co - localization analysis by epifluorescence microscopy of Sec24 - mRFP and de novo made UapA - GFP ( 8h derepressed transcription ) . Quantification of co - localization was made by calculating Pearson ' s Correlation Coefficient . For the statistical analysis one sample t - test was used . Notice the significant co - localization ( PCC = 0 . 61 ± 0 . 07 , * * * * P < 0 . 0001 ) . Due to the partially functional mRFP - tagged version of Sec24 ( evident in the growth test shown on the left panel ) , UapA trafficking is significantly delayed , reflected in the appearance of prominent labeling of uniform perinuclear ER . Scale bar : 5 μm . Biological / technical replicates : 3 / 5 . ( E ) Epifluorescence microscopy analysis of the subcellular localization of UapA - GFP under conditions where sec13 transcription is ab initio derepressed ( left panel ) or repressed by thiamine ( right panel ) . The inserts in the upper right corner in both panels reflect growth tests showing that Sec13 repression leads to arrest in growth and absence of colony formation . Scale bars : 5 μm . All images reflect practically identical results obtained in several experiments . Quantification : UapA - GFP PM / cytosolic intensity ratios are quantified when sec13 is derepressed ( - thi ) or repressed ( + thi ) , with mean values being 0 . 54 ± 0 . 02 and 0 . 31 ± 0 . 02 , respectively . There is a significant difference on UapA PM fluorescence intensity ( * * * * P < 0 . 0001 ) in the absence of the COPII outer coat protein ( statistical analysis as in B ) . Biological / technical replicates : 2 / 15 for each condition .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth test showing that Sec24 expression is essential for growth as its transcriptional repression by thiamine (+thi) leads to absence of colony formation, despite initial germination The strains shown are isogenic except for the sec24 locus. thiAp-sec24 signifies the strain where the endogenous sec24 promoter was replaced by the thiAp promoter.(B) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec24 transcription is ab initio derepressed (upper panel) or repressed by thiamine (lower panel). o/n (overnight) means addition of thiamine from the onset of germination. Germination of conidiospores takes place until full repression of Sec24 is achieved (10-12 h). Notice the total lack of PM-associated signal of UapA under conditions of Sec24 repression. Scale bar: 5 μm. Quantification: In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when sec24 is derepressed (-thi) or repressed (+thi). Mean PM/cytosolic intensity ratios are 0.55±0.02 and 0.32±0.02 respectively. To test the significance of differences, an unpaired t-test was performed, which verified the significant difference (****P<0.0001) in the presence of thiamine. Biological/technical replicates: 2/15 for each condition.(C) Epifluorescence microscopy analysis of the subcellular localization of the apical marker chitin synthase (GFP-ChsB) under sec24 derepressed or repressed conditions. Notice the loss of apical depositioning and the concurrent labeling of cytoplasmic foci and membranous structures under repression conditions. Scale bar: 5 μm. Quantification: GFP-ChsB PM/cytosolic intensity ratios are plotted in cases where sec24 is derepressed (-thi) or repressed (+thi). Mean values are 0.56±0.03 and 0.12±0.02 respectively. The fluorescence intensity of GFP-ChsB is statistically lower (****P<0.0001) when the expression of sec24 is repressed (statistical analysis as in B). Biological/technical replicates: 2/15 for each condition.(D) Co-localization analysis by epifluorescence microscopy of Sec24-mRFP and de novo made UapA-GFP (8h derepressed transcription). Quantification of co-localization was made by calculating Pearson's Correlation Coefficient. For the statistical analysis one sample t-test was used. Notice the significant co-localization (PCC=0.61±0.07, ****P<0.0001). Due to the partially functional mRFP-tagged version of Sec24 (evident in the growth test shown on the left panel), UapA trafficking is significantly delayed, reflected in the appearance of prominent labeling of uniform perinuclear ER. Scale bar: 5 μm. Biological/technical replicates: 3/5.(E) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec13 transcription is ab initio derepressed (left panel) or repressed by thiamine (right panel). The inserts in the upper right corner in both panels reflect growth tests showing that Sec13 repression leads to arrest in growth and absence of colony formation. Scale bars: 5 μm. All images reflect practically identical results obtained in several experiments. Quantification: UapA-GFP PM/cytosolic intensity ratios are quantified when sec13 is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.31±0.02, respectively. There is a significant difference on UapA PM fluorescence intensity (****P<0.0001) in the absence of the COPII outer coat protein (statistical analysis as in B). Biological/technical replicates: 2/15 for each condition.


Figure 5 ( A ) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA - GFP , after 6 - 8 hours of initiation of transcription in strains where the expression of RabE , AP - 1σ or ClaH ( thiAp - rabE , thiAp - ap1σ or thiAp - claH ) has been repressed ab initio ( o / n ) by addition of thiamine . Notice that when rabE or ap1σ is repressed UapA - GFP still reaches the PM . In contrast , repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network . Scale bars : 5 μm . Quantification : UapA - GFP PM / cytosolic intensity ratios are plotted on the right . Mean ratios for wild - type , thiAp - rabE , thiAp - ap1σ and thiAp - claH are 0 . 55 ± 0 . 02 , 0 . 53 ± 0 . 03 , 0 . 54 ± 0 . 02 and 0 . 29 ± 0 . 02 respectively . For the statistical analysis , Tukey ' s Multiple Comparison test was performed ( One - way ANOVA ) . After 6 - 8h of transcriptional derepression , UapA - GFP fluorescence to the PM does not change when rabE or ap1σ are repressed . On the other hand , in the absence of ClaH , UapA does not reach the PM , as its fluorescence intensity there is statistically lower ( * * * * P < 0 . 0001 ) in comparison to that of the wild - type strain . Biological / technical replicates : 2 / 15 for each strain . ( B ) In a similar experiment , the apical polarized localization of de novo made SynA , used as a control of conventional secretion , is shown to be abolished in all three strains where RabE , AP - 1σ or ClaH are repressed . Scale bars : 5 μm . mCherry - SynA PM / cytosolic intensity ratios are plotted with values being 0 . 52 ± 0 . 02 for wild - type , 0 . 14 ± 0 . 02 for thiAp - rabE , 0 . 24 ± 0 . 02 for thiAp - ap1σ and 0 . 16 ± 0 . 02 for thiAp - claH . The statistical evaluation was performed as in ( A ) . There is a significant difference ( * * * * P < 0 . 0001 ) of mCherry - SynA fluorescence in the apical PM membrane between the wild - type and the three strains lacking the post - Golgi proteins . Biological / technical replicates : 2 / 15 for each strain . ( C ) Co - localization analysis and relevant quantification of strains co - expressing de novo made UapA - GFP ( 120 min after initiation of transcription ) with RabE - mRFP , AP - 1 - mRFP or ClaH - mRFP . Scale bars : 2 μm . Quantification by calculating Pearson ' s Correlation Coefficient ( PCC ) shows clear non - colocalization of UapA with all the Post - Golgi markers tested , as confirmed by one sample t - test ( PCC = 0 . 31 ± 0 . 08 , 0 . 29 ± 0 . 04 or 0 . 07 ± 0 . 01 , with * * * * P < 0 . 0001 , for RabE , AP - 1 or ClaH , respectively ) . Biological / technical replicates : 3 / 7 for each strain .

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

Figure 5(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA-GFP, after 6-8 hours of initiation of transcription in strains where the expression of RabE, AP-1σ or ClaH (thiAp-rabE, thiAp-ap1σ or thiAp-claH) has been repressed ab initio (o/n) by addition of thiamine. Notice that when rabE or ap1σ is repressed UapA-GFP still reaches the PM. In contrast, repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network. Scale bars: 5 μm. Quantification: UapA-GFP PM/cytosolic intensity ratios are plotted on the right. Mean ratios for wild-type, thiAp-rabE, thiAp-ap1σ and thiAp-claH are 0.55±0.02, 0.53±0.03, 0.54±0.02 and 0.29±0.02 respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). After 6-8h of transcriptional derepression, UapA-GFP fluorescence to the PM does not change when rabE or ap1σ are repressed. On the other hand, in the absence of ClaH, UapA does not reach the PM, as its fluorescence intensity there is statistically lower (****P<0.0001) in comparison to that of the wild-type strain. Biological/technical replicates: 2/15 for each strain.(B) In a similar experiment, the apical polarized localization of de novo made SynA, used as a control of conventional secretion, is shown to be abolished in all three strains where RabE, AP-1σ or ClaH are repressed. Scale bars: 5 μm. mCherry-SynA PM/cytosolic intensity ratios are plotted with values being 0.52±0.02 for wild-type, 0.14±0.02 for thiAp-rabE, 0.24±0.02 for thiAp-ap1σ and 0.16±0.02 for thiAp-claH. The statistical evaluation was performed as in (A). There is a significant difference (****P<0.0001) of mCherry-SynA fluorescence in the apical PM membrane between the wild-type and the three strains lacking the post-Golgi proteins. Biological/technical replicates: 2/15 for each strain.(C) Co-localization analysis and relevant quantification of strains co-expressing de novo made UapA-GFP (120 min after initiation of transcription) with RabE-mRFP, AP-1-mRFP or ClaH-mRFP. Scale bars: 2 μm. Quantification by calculating Pearson's Correlation Coefficient (PCC) shows clear non-colocalization of UapA with all the Post-Golgi markers tested, as confirmed by one sample t-test (PCC=0.31±0.08, 0.29±0.04 or 0.07±0.01, with ****P<0.0001, for RabE, AP-1 or ClaH, respectively). Biological/technical replicates: 3/7 for each strain.


Figure 6 ( A ) Time course of treatment with the actin polymerization drug latrunculin B for 2 , 5 , 10 , 30 or 50 minutes of a strain expressing neosynthesized UapA - GFP under conditions of derepression compared to an untreated strain included as control ( 150 min ) . In all cases latrunculin B was added at 100 min of UapA derepression , so that total time of UapA - GFP expression was 102 , 105 , 110 , 130 , or 150 minutes in the different samples . Notice the abolishment of sorting of UapA to the PM after 130 or 150 min of expression when latrunculin B was present for the last 30 or 50 min , respectively . Scale bar : 5 μm . ( B ) Time course of treatment of strains expressing neosynthesized alcAp - UapA - GFP and mCherry - TubA with the anti - microtubule drug benomyl for 5 , 55 , or 95 minutes . In all cases benomyl was added at 95 min of UapA derepression , so that total time of UapA - GFP expression was 100 , 150 or 190 minutes Benomyl abolished the thread - like appearance microtubules in all samples added , evident by the diffuse cytoplasmic signal of mCherry - TubA . Notice that UapA reaches normally the PM ( at 190 min of derepression ) , similarly to the untreated control ( right panel ) . Scale bar : 5 μm . ( C ) Subcellular localization of neosynthesized alcAp - GFP - ChsB in the absence ( left panel ) or presence ( ≈ 40 min ) of benomyl ( middle panel ) or latrunculin B ( right panel ) , after ≈ 2h of derepression . Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases . Scale bars : 5 μm . ( D ) Effect of latrunculin B or benomyl on COPII vesicle formation , followed through the subcellular localization Sec24 - GFP . Notice that 40 min of latrunculin B led to loss of the wild - type punctuate appearance of Sec24 cytoplasmic puncta and the appearance of a diffuse fluorescent haze in the cytoplasm . In contrast , benomyl treatment did not affect the wild - type punctuate localization of Sec24 . Scale bar : 5 μm .

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

Figure 6(A) Time course of treatment with the actin polymerization drug latrunculin B for 2, 5, 10, 30 or 50 minutes of a strain expressing neosynthesized UapA-GFP under conditions of derepression compared to an untreated strain included as control (150 min). In all cases latrunculin B was added at 100 min of UapA derepression, so that total time of UapA-GFP expression was 102, 105, 110, 130, or 150 minutes in the different samples. Notice the abolishment of sorting of UapA to the PM after 130 or 150 min of expression when latrunculin B was present for the last 30 or 50 min, respectively. Scale bar: 5 μm.(B) Time course of treatment of strains expressing neosynthesized alcAp-UapA-GFP and mCherry-TubA with the anti-microtubule drug benomyl for 5, 55, or 95 minutes. In all cases benomyl was added at 95 min of UapA derepression, so that total time of UapA-GFP expression was 100, 150 or 190 minutes Benomyl abolished the thread-like appearance microtubules in all samples added, evident by the diffuse cytoplasmic signal of mCherry-TubA. Notice that UapA reaches normally the PM (at 190 min of derepression), similarly to the untreated control (right panel). Scale bar: 5 μm.(C) Subcellular localization of neosynthesized alcAp-GFP-ChsB in the absence (left panel) or presence (≈40 min) of benomyl (middle panel) or latrunculin B (right panel), after ≈2h of derepression. Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases. Scale bars: 5 μm.(D) Effect of latrunculin B or benomyl on COPII vesicle formation, followed through the subcellular localization Sec24-GFP. Notice that 40 min of latrunculin B led to loss of the wild-type punctuate appearance of Sec24 cytoplasmic puncta and the appearance of a diffuse fluorescent haze in the cytoplasm. In contrast, benomyl treatment did not affect the wild-type punctuate localization of Sec24. Scale bar: 5 μm.


Figure 7 ( A ) Subcellular localization of de novo made alcAp - GFP - SsoA in the PM in a single hypha . Notice the ER - like membranous network ( white arrows ) and several cytosolic puncta labelled before SsoA reaches eventually the PM . Scale bar : 5 μm . ( B ) Subcellular localization of neosynthesized UapA - GFP ( 8 hours after transcriptional depression ) while ssoA transcription is ab initio repressed . Notice the localization of UapA in the ER - membranous network , often at very close proximity with the PM . Scale bar : 5 μm . In the scatter plot on the right , UapA - GFP PM / cytosolic intensity ratios are quantified when ssoA is derepressed ( - thi ) or repressed ( + thi ) , with mean values being 0 . 54 ± 0 . 02 and 0 . 30 ± 0 . 02 respectively . There is statistically lower fluorescence intensity to the PM ( * * * * P < 0 . 0001 ) when SsoA is absent from the cell , as confirmed by an unpaired t - test . Biological / technical replicates : 2 / 15 for each condition .

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 O O O O O O O O O O O O O O O O O O O O O O O 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 7(A) Subcellular localization of de novo made alcAp-GFP-SsoA in the PM in a single hypha. Notice the ER-like membranous network (white arrows) and several cytosolic puncta labelled before SsoA reaches eventually the PM. Scale bar: 5 μm.(B) Subcellular localization of neosynthesized UapA-GFP (8 hours after transcriptional depression) while ssoA transcription is ab initio repressed. Notice the localization of UapA in the ER-membranous network, often at very close proximity with the PM. Scale bar: 5 μm. In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when ssoA is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.30±0.02 respectively. There is statistically lower fluorescence intensity to the PM (****P<0.0001) when SsoA is absent from the cell, as confirmed by an unpaired t-test. Biological/technical replicates: 2/15 for each condition.


Figure 8 ( A ) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp - AzgA - GFP and alcAp - FurA - GFP , after 6 - 8 hours transcriptional derepression in strains where the expression of Sec24 , SedV , GeaA , HypB , RabE , AP - 1σ and SsoA was repressed ab initio ( o / n ) by addition of thiamine . Images showing that sorting of AzgA or FurA to the PM does not require early ( SedV , GeaA ) , late ( HypB ) or post - Golgi ( RabE , AP - 1σ ) key proteins . On the other hand , COPII coat protein Sec24 and PM t - SNARE SsoA are essential for proper localization of the two transporters to the PM . Scale bar : 5 μm . ( B ) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters ( see text for details ) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated . Notice that addition of Benomyl , has no effect on translocation of AzgA and FurA to the PM , whereas Latrunculin B abolishes their PM localization , as seen also for UapA . Scale bar : 5 μm .

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 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 8(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm.(B) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters (see text for details) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated. Notice that addition of Benomyl, has no effect on translocation of AzgA and FurA to the PM, whereas Latrunculin B abolishes their PM localization, as seen also for UapA. Scale bar: 5 μm.


Figure 1CD4 and CD8 T cells were stimulated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 beads as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . Blots depict one representative donor out of three . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 ; and human IFNβ ( top ) and IP10 levels ( bottom ) in the supernatant were analyzed by ELISA at the indicated time points . Data panels depict mean ± SD of biological duplicates from three independent donors . CD4 T cells were pre - treated with cycloheximide ( 2 μg / ml ) for 2 h , then treated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 ( 3 / 28 ) for 4 h , then analyzed by quantitative PCR for IFNB1 and OASL expression . Data are depicted as mean + SEM of 3 independent donors ( for cGAMP and αCD3 / CD28 treated cells without CHX only 2 data points were obtained ) . Statistics were determined by a two - way ANOVA on log - transformed data using Šidák correction for multiple testing : * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1CD4 and CD8 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28 beads as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. Blots depict one representative donor out of three.CD4 T cells were stimulated with cGAMP (20 μg/ml) and αCD3/CD28; and human IFNβ (top) and IP10 levels (bottom) in the supernatant were analyzed by ELISA at the indicated time points. Data panels depict mean ± SD of biological duplicates from three independent donors.CD4 T cells were pre-treated with cycloheximide (2 μg/ml) for 2 h, then treated with cGAMP (20 μg/ml) and αCD3/CD28 (3/28) for 4 h, then analyzed by quantitative PCR for IFNB1 and OASL expression. Data are depicted as mean + SEM of 3 independent donors (for cGAMP and αCD3/CD28 treated cells without CHX only 2 data points were obtained). Statistics were determined by a two-way ANOVA on log-transformed data using Šidák correction for multiple testing: ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant.


Figure 2CD4 T cells were stimulated with the indicated stimuli for 4 h . Lysates were analyzed by immunoblotting . One representative donor out of three is shown . CRISPR / Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h . Human IFNβ levels were analyzed by ELISA . Data are depicted as mean + SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Dunnett correction for multiple testing : * * * * p < 0 . 0001 ; ns , not significant .

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Figure 2CD4 T cells were stimulated with the indicated stimuli for 4 h. Lysates were analyzed by immunoblotting. One representative donor out of three is shown.CRISPR/Cas9 targeted CD4 T cells for the indicated genes were treated with the indicated stimuli for 48 h. Human IFNβ levels were analyzed by ELISA. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ****p < 0.0001; ns, not significant.


Figure 3CD4 T cells were pre - treated with Dasatinib ( 100 , 10 , 1 and 0 . 1 nM ) , Tacrolimus ( 1000 , 100 , 10 and 1 nM ) , TPCA - 1 ( 4 , 2 , 1 and 0 . 5 μM ) and Torin1 ( 50 , 5 , 0 . 5 and 0 . 05 nM ) for 30 min , then treated with cGAMP ( 20 μg / ml ) and αCD3 / CD28 for 16 h . Ηuman IFNβ levels in the supernatant were analyzed by ELISA . Data panels depict mean of biological duplicates from three independent donors . CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA - 1 for 4 h . Cell lysates were analyzed by immunoblotting . One representative donor out of two is shown . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and additional stimuli as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . One representative donor out of two is shown . CD4 T cells were stimulated with cGAMP ( 20 μg / ml ) and additional stimuli as indicated . Cell lysates after 4 h of stimulation were analyzed by immunoblotting . One representative donor out of two is shown .

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

Figure 3CD4 T cells were pre-treated with Dasatinib (100, 10, 1 and 0.1 nM), Tacrolimus (1000, 100, 10 and 1 nM), TPCA-1 (4, 2, 1 and 0.5 μM) and Torin1 (50, 5, 0.5 and 0.05 nM) for 30 min, then treated with cGAMP (20 μg/ml) and αCD3/CD28 for 16 h. Ηuman IFNβ levels in the supernatant were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors.CD4 T cells were treated with cGAMP and indicated stimuli together with increasing concentrations of TPCA-1 for 4 h. Cell lysates were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown.


Figure 4Freshly isolated CD4 T cells were treated with cGAMP , αCD3 / CD28 beads and ABT737 / S63845 ( A / S ) for 48 h and analyzed by flow cytometry . Treatment induced cell death was assessed by Annexin V and propidium iodide staining . Data are depicted as mean + SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Dunnett correction for multiple testing : * * * p < 0 . 001 ; * p < 0 . 05 ; ns , not significant . CD4 T cells were treated with indicated concentrations of cGAMP for 18 h , and ABT737 / S63845 for 2 h . Lysates and supernatant precipitations were analyzed by immunoblotting . One representative donor out of two is shown . CRISPR / Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry . Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three . Digits indicate the percentage of the parental population ( single events without subcellular debris ) .

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Figure 4Freshly isolated CD4 T cells were treated with cGAMP, αCD3/CD28 beads and ABT737/S63845 (A/S) for 48 h and analyzed by flow cytometry. Treatment induced cell death was assessed by Annexin V and propidium iodide staining. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ***p < 0.001; *p < 0.05; ns, not significant.CD4 T cells were treated with indicated concentrations of cGAMP for 18 h, and ABT737/S63845 for 2 h. Lysates and supernatant precipitations were analyzed by immunoblotting. One representative donor out of two is shown.CRISPR/Cas9 targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry. Each panel shows propidium iodide and Annexin V intensity for one representative donor out of three. Digits indicate the percentage of the parental population (single events without subcellular debris).


Figure 5CellTrace Violet ( CTV ) stained , freshly isolated CD4 and CD8 T cells were activated with αCD3 / CD28 beads , stimulated with cGAMP for 96 h and analyzed by flow cytometry . Each panel shows profiles from activated ( black ) , cGAMP - treated ( green ) and resting ( light gray ) single and live cells from one representative donor . Freshly isolated CD4 T cells were treated with cGAMP ( 40 μg / ml ) and αCD3 / CD28 ( 3 / 28 ) for 48 h and analyzed by Seahorse XF mito stress test . Data are depicted as mean ± SEM of 3 independent donors . Statistics indicate significance by two - way ANOVA with Šidák correction for multiple testing : * * p < 0 . 01 ; * p < 0 . 05 ; ns , not significant .

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Figure 5CellTrace Violet (CTV) stained, freshly isolated CD4 and CD8 T cells were activated with αCD3/CD28 beads, stimulated with cGAMP for 96 h and analyzed by flow cytometry. Each panel shows profiles from activated (black), cGAMP-treated (green) and resting (light gray) single and live cells from one representative donor.Freshly isolated CD4 T cells were treated with cGAMP (40 μg/ml) and αCD3/CD28 (3/28) for 48 h and analyzed by Seahorse XF mito stress test. Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; *p < 0.05; ns, not significant.


Figure 1Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3 - / - naïve mESCs . DNA counterstain is Hoechst . Dashed white lines indicate the embryonic Gata6 : : mCherry negative part . Scale bar : 10 μm .

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Figure 1Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3-/- naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm.


Figure 2Nanog > GFP and Gata6 : : mCherry fluorescence intensities of WT and Hdac3 - / - Fraction of Gata6 : : mCherry positive cells in indicated genotypes and conditions after 4d Jak ( i ) blocks LIF , LDN19 BMP4 and PD03 FGF signaling . Average and SD of at least two independent clones . Quantification of spatial lineage segregation in spheroids . Representative spheroid segmentations of indicated genotypes Scale bar : 25 μm .

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Figure 2Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 4d Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones.Quantification of spatial lineage segregation in spheroids. Representative spheroid segmentations of indicated genotypes Scale bar: 25 μm.


Figure 3Scatter plots of log2 fold change ( FC ) gene expression changes ( ∆ mRNA ) between naïve Hdac3 - / - or Dax1 - / - mutants and WT mESCs ( left ) , between Nanog > GFP - expressing Hdac3 - / - or Dax1 - / - mutants and WT cells ( middle ) , and between Gata6 : : mCherry - expressing Hdac3 - / - or Dax1 - / - mutants and Nanog > GFP - expressing WT cells ( right ) . Cluster 5 genes are colored and selected PrE TFs labeled .

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Figure 3Scatter plots of log2 fold change (FC) gene expression changes (∆mRNA) between naïve Hdac3-/- or Dax1-/- mutants and WT mESCs (left), between Nanog>GFP-expressing Hdac3-/- or Dax1-/- mutants and WT cells (middle), and between Gata6::mCherry-expressing Hdac3-/- or Dax1-/- mutants and Nanog>GFP-expressing WT cells (right). Cluster 5 genes are colored and selected PrE TFs labeled.


Figure 4Expression of PrE markers relative to Dax1 - / - cells ( B ) , and fraction of Gata6 : : mCherry positive cells after 3d in SLRA ( C ) in indicated genotypes . Average and SD of three independent clones .

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Figure 4Expression of PrE markers relative to Dax1-/- cells (B), and fraction of Gata6::mCherry positive cells after 3d in SLRA (C) in indicated genotypes. Average and SD of three independent clones.


Figure 5Genome - browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants , and of ChIPseq signals of indicated TFs and H3K27ac in E6 . 5 VE and EPI . The grey area indicates enh - 45 . FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA ( D ) and in 2iL ( E ) . Average and SD of at least three independent clones .

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Figure 5Genome-browser view of the Gata6 locus showing accessibility and chromatin mark changes in indicated mutants, and of ChIPseq signals of indicated TFs and H3K27ac in E6.5 VE and EPI. The grey area indicates enh-45.FC of PrE and naïve pluripotency markers relative to WT cells in 2iL of indicated genotypes after 3d in SLRA (D) and in 2iL (E). Average and SD of at least three independent clones.


Figure 1Heatmap showing the normalized expression of marker genes for the different clusters ( Methods ) . Expression is normalized by the maximal expression among all cell types .

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Figure 1Heatmap showing the normalized expression of marker genes for the different clusters (Methods). Expression is normalized by the maximal expression among all cell types.


Figure 2Top - left - tSNE plot of the Seurat clusters , boxes demarcate compared clusters . Dashed boxes and labels indicate the cell clusters that are compared in panels A - C . ( A ) Volcano plot of differential gene expression ( DGE ) between liver vascular endothelial cells in the tumor and non - tumor samples . ( B ) Volcano plot of DGE between mononuclear phagocytes in the malignant and non - malignant samples . ( C ) Volcano plot of DGE between T cells in the tumor and non - tumor samples . ( D ) DGE analysis between tumor mononuclear phagocytes classified by cancer type ( cholangiocarcinoma in dark purple and metastasis in light purple ) . Wilcoxon rank - sum tests were used to generate p - values , Benjamini - Hochberg multiple hypotheses correction was used to compute q - values . Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two clusters .

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Figure 2Top-left - tSNE plot of the Seurat clusters, boxes demarcate compared clusters. Dashed boxes and labels indicate the cell clusters that are compared in panels A-C. (A) Volcano plot of differential gene expression (DGE) between liver vascular endothelial cells in the tumor and non-tumor samples. (B) Volcano plot of DGE between mononuclear phagocytes in the malignant and non-malignant samples. (C) Volcano plot of DGE between T cells in the tumor and non-tumor samples. (D) DGE analysis between tumor mononuclear phagocytes classified by cancer type (cholangiocarcinoma in dark purple and metastasis in light purple). Wilcoxon rank-sum tests were used to generate p-values, Benjamini-Hochberg multiple hypotheses correction was used to compute q-values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two clusters.


Figure 3Key marker genes for the four mesenchymal clusters ( RBP1 , COL1A1 , RGS5 , and MYH11 ) . Light gray dots denote cells originating from non - tumor samples . Dark gray dots denote cells originating from the tumor samples . Left - Representative smFISH image of patient p1 stained for RGS5 and COL1A1 showing distinct spatial localization of CAFs and Pericytes . Scale bar 10µm . Dashed lines mark the shortest distance of cells ( 2a ) and ( 2b ) from the cell ( 1 ) . Middle - zoom - in of ( 1 ) from left panel , showing a blood vessel like structure formed by endothelial cells marked by PDGFB ( magenta ) wrapped by pericytes marked by RGS5 ( green ) . Dashed lines are two consecutive cell layers of endothelial cells and pericytes . Scale bar 2 . 5µm . Right - zoom - in of ( 2a and b ) from the left panel , showing two distant CAFs expressing high COL1A1 signal but not RGS5 . DAPI used for nuclei staining . Scale bar 2 . 5µm . Violin plot of the distance from blood vessels of low / high RGS5 expressing cells ( n = 358 and n = 360 respectively ) and low / high COL1A1 expressing cells ( n = 359 and n = 359 respectively ) . ' p ' is the p - value determined by Wilcoxon rank - sum test . Empty circles are the medians over all repeats .

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Figure 3Key marker genes for the four mesenchymal clusters (RBP1, COL1A1, RGS5, and MYH11). Light gray dots denote cells originating from non-tumor samples. Dark gray dots denote cells originating from the tumor samples. Left - Representative smFISH image of patient p1 stained for RGS5 and COL1A1 showing distinct spatial localization of CAFs and Pericytes. Scale bar 10µm. Dashed lines mark the shortest distance of cells (2a) and (2b) from the cell (1). Middle - zoom-in of (1) from left panel, showing a blood vessel like structure formed by endothelial cells marked by PDGFB (magenta) wrapped by pericytes marked by RGS5 (green). Dashed lines are two consecutive cell layers of endothelial cells and pericytes. Scale bar 2.5µm. Right - zoom-in of (2a and b) from the left panel, showing two distant CAFs expressing high COL1A1 signal but not RGS5. DAPI used for nuclei staining. Scale bar 2.5µm.Violin plot of the distance from blood vessels of low/high RGS5 expressing cells (n=358 and n= 360 respectively) and low/high COL1A1 expressing cells (n=359 and n= 359 respectively). 'p' is the p-value determined by Wilcoxon rank-sum test. Empty circles are the medians over all repeats.


Figure 4Dot - plot of selected genes highlight shared interaction motifs between different clusters colored by max normalized expression for each genes across all clusters in ( A ) . For each gene , dot size represents the fraction of positive cells for each cluster .

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Figure 4Dot-plot of selected genes highlight shared interaction motifs between different clusters colored by max normalized expression for each genes across all clusters in (A). For each gene, dot size represents the fraction of positive cells for each cluster.


Figure 5H & E staining of malignant and non - malignant sites from patient p4 , separated by the white dashed line . Scale bar is 100µm . Non - malignant human liver lobule from patient p4 used for LCM stained with HistoGene Staining Solution ( Methods ) . Top - sequential zones from the central vein ( cv ) to the portal node ( pn ) before microdissection . LCM captured zones marked by the black dashed line . White dashed line marks the boundaries of NPC ( non - parenchymal cells ) located in the pn and not included in the analysis . Bottom - Tissue after microdissection . Scale bar is 100µm . Zonation of human hepatocytes . Left - Max normalized expression over the reconstructed six lobule layers ( L1 to L6 ) , sorted by center of mass from the central vein ( cv ) to the portal node ( pn ) . Right - selected pericentrally ( top ) , and periportally ( bottom ) zonated genes . Blue patches are standard errors of the mean . Zonation profiles of selected genes from human ( blue ) and mouse ( green ) . Each profile is normalized by the maximal expression across zones . Patches are standard errors of the mean .

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Figure 5H&E staining of malignant and non-malignant sites from patient p4, separated by the white dashed line. Scale bar is 100µm. Non-malignant human liver lobule from patient p4 used for LCM stained with HistoGene Staining Solution (Methods). Top - sequential zones from the central vein (cv) to the portal node (pn) before microdissection. LCM captured zones marked by the black dashed line. White dashed line marks the boundaries of NPC (non-parenchymal cells) located in the pn and not included in the analysis. Bottom - Tissue after microdissection. Scale bar is 100µm.Zonation of human hepatocytes. Left - Max normalized expression over the reconstructed six lobule layers (L1 to L6), sorted by center of mass from the central vein (cv) to the portal node (pn). Right - selected pericentrally (top), and periportally (bottom) zonated genes. Blue patches are standard errors of the mean.Zonation profiles of selected genes from human (blue) and mouse (green). Each profile is normalized by the maximal expression across zones. Patches are standard errors of the mean.


Figure 6tissue sections from patients p1 and p3 stained with HistoGene Staining Solution . Laser captured zones are marked with dashed black line including : tumor core ( tc ) , the border between the tumor and the fibrotic regions ( ftb ) , tumor islets within the fibrotic regions ( ti ) and fibrotic zones ( fz ) in panel ( A ) , and tumor border ( tb ) in panel ( B ) . Small images in ( A ) are magnifications of the corresponding zones in the large image for the left . White dashed line in ( B ) marking the non - malignant liver ( nml ) border . Scale bars in all panels are 100µm .

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Figure 6tissue sections from patients p1 and p3 stained with HistoGene Staining Solution. Laser captured zones are marked with dashed black line including: tumor core (tc), the border between the tumor and the fibrotic regions (ftb), tumor islets within the fibrotic regions (ti) and fibrotic zones (fz) in panel (A), and tumor border (tb) in panel (B). Small images in (A) are magnifications of the corresponding zones in the large image for the left. White dashed line in (B) marking the non-malignant liver (nml) border. Scale bars in all panels are 100µm.


Figure 1 ( A ) siRNA knockdown of Tollip in SH - SY5Y cells was performed over 72 hours , with cells reverse transfected with siRNA oligos specific for Tollip ( or mock ) for 24 hours before a second transfection was performed for a further 48 hours . Knockdown was confirmed by western blotting . ( B ) After knockdown of Tollip , SH - SY5Y cells were treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or 25 μM Antimycin A ( AA ) alone for 2 hours , then fixed and stained for PDH E2 / E3bp ( green ) , TOM20 ( red ) and nuclei ( blue ) . Images were captured using widefield immunofluorescence microscopy . Zoom insets represent 4x magnification and arrowheads indicate TOM20 - positive MDVs . ( C , D ) The number of TOM20 + ve / PDH - ve MDVs ( C ) or PDH + ve / TOM20 - ve MDVs ( D ) per cell were quantified from immunofluorescence images captured ( E ) SH - SY5Y cells were transfected with GFP - labelled Tollip for 24 hours then treated with AO , AO / 100 μM bafilomycin A1 ( AO / BfnA1 ) or AA alone for 2 hours prior to fixation . Cells were then immunostained for GFP ( green ) , PDH E2 / E3bp ( red ) and TOM20 ( blue ) and imaged by widefield immunofluorescence microscopy . GFP - Tollip was observed on TOM20 + ve / PDH - ve MDVs ( arrowheads ) . Zoom insets represent 7x magnification . ( F ) Images captured using super - resolution radial fluctuations ( SRRF ) microscopy showing both TOM20 ( arrowheads ) and PDH ( arrows ) MDVs in SH - SY5Y cells after Tollip siRNA knockdown . Left side images are the first in each of the original widefield image stacks . Scale bar for zoom images is 0 . 5 μm .

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Figure 1(A) siRNA knockdown of Tollip in SH-SY5Y cells was performed over 72 hours, with cells reverse transfected with siRNA oligos specific for Tollip (or mock) for 24 hours before a second transfection was performed for a further 48 hours. Knockdown was confirmed by western blotting.(B) After knockdown of Tollip, SH-SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM Antimycin A (AA) alone for 2 hours, then fixed and stained for PDH E2/E3bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4x magnification and arrowheads indicate TOM20-positive MDVs.(C, D) The number of TOM20+ve/PDH-ve MDVs (C) or PDH+ve/TOM20-ve MDVs (D) per cell were quantified from immunofluorescence images captured(E) SH-SY5Y cells were transfected with GFP-labelled Tollip for 24 hours then treated with AO, AO/100 μM bafilomycin A1 (AO/BfnA1) or AA alone for 2 hours prior to fixation. Cells were then immunostained for GFP (green), PDH E2/E3bp (red) and TOM20 (blue) and imaged by widefield immunofluorescence microscopy. GFP-Tollip was observed on TOM20+ve/PDH-ve MDVs (arrowheads). Zoom insets represent 7x magnification.(F) Images captured using super-resolution radial fluctuations (SRRF) microscopy showing both TOM20 (arrowheads) and PDH (arrows) MDVs in SH-SY5Y cells after Tollip siRNA knockdown. Left side images are the first in each of the original widefield image stacks. Scale bar for zoom images is 0.5 μm.


Figure 2 ( A ) TOM20 - positive MDV formation was induced in SH - SY5Y by siRNA knockdown of Tollip . MDVs were defined as TOM20 + ve / Cytochrome c ( Cyt c ) - ve via immunofluorescence microscopy , which indicated some colocalisation with Parkin ( arrowheads ) . ( B ) SH - SY5Y cells were transfected with GFP - Tollip for 24 hours , then treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A1 ( BfnA1 ) for 2 hours prior to fixation and immunostaining . Antibodies specific to GFP ( green ) , Parkin ( red ) and ubiquitin ( blue ) were used . C ) GFP - Tollip / Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO / BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell , across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ) . Results are represented as a percentage of Tollip puncta positive for Parkin . ( D ) SH - SY5Y cells expressing GFP - Tollip were immunostained for GFP ( green ) , Parkin ( red ) , and Rab7a ( blue ) . Arrowheads indicate areas of colocalisation between all three markers . ( E - F ) SH - SY5Y cells were transfected with GFP - Tollip R78A or CUE mutant for 24 hours , then treated with AO or AO / BfnA1 for 2 hours prior to fixation and immunostaining ( G ) The percentage of GFP - Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0162 , * * p = 0 . 0034 , * * * * p < 0 . 0001 ) . ( H ) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0382 , * * p = 0 . 0068 ) . ( I ) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0232 , * * p = 0 . 0062 ) . ( J ) The percentage of GFP - Tollip / Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP , Parkin , and ubiquitin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * * p = 0 . 0070 ) .

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Figure 2(A) TOM20-positive MDV formation was induced in SH-SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20+ve/Cytochrome c (Cyt c)-ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads).(B) SH-SY5Y cells were transfected with GFP-Tollip for 24 hours, then treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 hours prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used.C) GFP-Tollip/Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3-6 GFP-transfected cells/condition, per experiment (n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin.(D) SH-SY5Y cells expressing GFP-Tollip were immunostained for GFP (green), Parkin (red), and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers.(E-F) SH-SY5Y cells were transfected with GFP-Tollip R78A or CUE mutant for 24 hours, then treated with AO or AO/BfnA1 for 2 hours prior to fixation and immunostaining(G) The percentage of GFP-Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/ condition, per experiment (n = 3; *p=0.0162, **p=0.0034, ****p<0.0001).(H) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0382, **p=0.0068).(I) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0232, **p=0.0062).(J) The percentage of GFP-Tollip/Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP, Parkin, and ubiquitin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; **p=0.0070).


Figure 3 ( A ) HeLa cells expressing mycBioID - Tollip and HA - Parkin were either left untreated or treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A ( BfnA1 ) for 6 hours in the presence of biotin . Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins . Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS - PAGE and membranes probed with specific antibodies . Immunoblotting for Tom1 and mycBioID - Tollip were used as positive controls . Cells cultured in media lacking biotin were used as a negative control to assess background levels . Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions . ( B ) HeLa cells expressing mycBioID alone and HA - Parkin were used to confirm the specificity of the Tollip - Parkin interaction . ( C ) Hela cells co - expressing GFP - Tollip and HA - Parkin were either left untreated or treated with AO for 2 hours prior to lysis . Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP . ( D ) HeLa cells expressing mycBioID - Tollip containing the CUE domain mutation ( CUEmut ) and HA - Parkin were subjected to biotinylation and streptavidin pulldowns , followed by western blot analysis . ( E ) HeLa wild - type ( WT ) , Tom1 KO and ATG5 KO cells expressing mycBioID - Tollip and HA - Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins .

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Figure 3(A) HeLa cells expressing mycBioID-Tollip and HA-Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 hours in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS-PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID-Tollip were used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions.(B) HeLa cells expressing mycBioID alone and HA-Parkin were used to confirm the specificity of the Tollip-Parkin interaction.(C) Hela cells co-expressing GFP-Tollip and HA-Parkin were either left untreated or treated with AO for 2 hours prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP.(D) HeLa cells expressing mycBioID-Tollip containing the CUE domain mutation (CUEmut) and HA-Parkin were subjected to biotinylation and streptavidin pulldowns, followed by western blot analysis.(E) HeLa wild-type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID-Tollip and HA-Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins.


Figure 4 ( A ) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours , then fixed and stained with antibodies specific to TOM20 ( green ) and PDH E2 / E3bp ( red ) . Nuclei are labelled with Hoechst ( blue ) . Zoom insets represent 5x magnification and arrowheads indicate TOM20 - positive MDVs . ( B ) Knockdowns were confirmed for both Parkin and Tom1 by western blotting . ( C , D ) Quantification of MDVs , defined as TOM20 + ve / PDH - ve , ( C ) or PDH + ve / TOM20 - ve ( D ) per cell under steady - state conditions were performed ( n = 32 - 52 cells per condition from 3 independent experiments ) . ( E ) Western blot analysis of lysates harvested from SH - SY5Y wild - type ( empty vector ) and CRISPR generated Parkin KO cells . Immunoblot analysis was performed using antibodies against Parkin and GAPDH . ( F ) Immunofluorescence microscopy was performed on SH - SY5Y wild - type and Parkin KO cells either untreated or treated with AO for 2 hours . Cells were immunostained for TOM20 ( red ) , PDH E2 / E3bp ( blue ) , and LAMP1 ( green ) . Zoom insets represent 5x magnification . Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1 . Scale bar represents 10 µm . ( G ) Quantitation of TOM20 - positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20 - positive MDVs colocalising with LAMP1 . Results represent 3 independent experiments ( 28 - 53 cells / condition ) and statistical significance was determined using a Two - way ANOVA ( Sidak ) . ( H ) HeLa cells transiently transfected with HA - Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A ( AA ) for 2 hours , then fixed and stained with antibodies specific to HA , PDH and TOM20 . TOM20 + ve / PDH - ve MDVs were quantified in cells that were either expressing or not expressing HA - Parkin ( n = 12 - 14 cells per condition from 3 independent experiments ) . ( I ) Hela cells stably coexpressing BioID - Tollip and either HA - Parkin WT or Δ1 - 76 ( ΔUBL ) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns . Western blot analysis was performed on lysate inputs and streptavidin pulldowns ( Streptavidin PD ) using antibodies against the indicated proteins or epitope tags .

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Figure 4(A) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours, then fixed and stained with antibodies specific to TOM20 (green) and PDH E2/E3bp (red). Nuclei are labelled with Hoechst (blue). Zoom insets represent 5x magnification and arrowheads indicate TOM20-positive MDVs.(B) Knockdowns were confirmed for both Parkin and Tom1 by western blotting.(C, D) Quantification of MDVs, defined as TOM20+ve/PDH-ve, (C) or PDH+ve/TOM20-ve (D) per cell under steady-state conditions were performed (n = 32-52 cells per condition from 3 independent experiments).(E) Western blot analysis of lysates harvested from SH-SY5Y wild-type (empty vector) and CRISPR generated Parkin KO cells. Immunoblot analysis was performed using antibodies against Parkin and GAPDH.(F) Immunofluorescence microscopy was performed on SH-SY5Y wild-type and Parkin KO cells either untreated or treated with AO for 2 hours. Cells were immunostained for TOM20 (red), PDH E2/E3bp (blue), and LAMP1 (green). Zoom insets represent 5x magnification. Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1. Scale bar represents 10 µm.(G) Quantitation of TOM20-positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20-positive MDVs colocalising with LAMP1. Results represent 3 independent experiments (28-53 cells/condition) and statistical significance was determined using a Two-way ANOVA (Sidak).(H) HeLa cells transiently transfected with HA-Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A (AA) for 2 hours, then fixed and stained with antibodies specific to HA, PDH and TOM20. TOM20+ve/PDH-ve MDVs were quantified in cells that were either expressing or not expressing HA-Parkin (n = 12-14 cells per condition from 3 independent experiments).(I) Hela cells stably coexpressing BioID-Tollip and either HA-Parkin WT or Δ1-76 (ΔUBL) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns. Western blot analysis was performed on lysate inputs and streptavidin pulldowns (Streptavidin PD) using antibodies against the indicated proteins or epitope tags.


Figure 5 ( A - B ) siRNA knockdown of Tollip or Parkin in SH - SY5Y cells was performed over a 72 hour period , then 18 hours prior to treatment cells were transfected with GFP - 2xFYVE to label endocytic membranes . Cells were treated with 25 μM antimycin A ( AA ) for 2 hours then fixed and immunostained with antibodies specific to PDH E2 / E3bp ( red ) and TOM20 ( blue ) . Arrowheads denote TOM20 + ve / PDH - ve MDVs that colocalise with GFP - 2xFYVE and arrows denote TOM20 + ve / PDH - ve MDVs that do not . ( C ) Quantification of TOM20 + ve / PDH - ve MDVs that are positive for GFP - 2xFYVE are shown as a % of TOM20 MDVs positive for GFP , with each data point indicating 1 cell . MDV colocalisation were quantified by eye from images captured ( n = 20 - 22 cells per condition from 3 independent experiments ) . ( D ) SH - SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 ( green ) , PDH E2 / E3bp ( red ) , and TOM20 ( blue ) . Arrowheads indicate TOM20 + ve / PDH - ve MDVs positive for the early endosome marker EEA1 . ( E ) siRNA knockdown of Parkin was performed in SH - SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 ( green ) , Tollip ( red ) and Cytochrome c ( blue ) . We observed Tollip colocalisation with TOM20 + ve / PDH - ve MDVs ( denoted by arrowheads ) , which was still maintained following Parkin siRNA knockdown . ( F ) SH - SY5Y cells were transfected with GFP - Tollip and treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A ( BfnA1 ) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP ( green ) , Rab7a ( red ) and PDH E2 / E3bp ( blue ) . ( G ) Quantification of GFP - Tollip / Rab7a colocalisation using Pearson ' s correlation .

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Figure 5(A-B) siRNA knockdown of Tollip or Parkin in SH-SY5Y cells was performed over a 72 hour period, then 18 hours prior to treatment cells were transfected with GFP-2xFYVE to label endocytic membranes. Cells were treated with 25 μM antimycin A (AA) for 2 hours then fixed and immunostained with antibodies specific to PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs that colocalise with GFP-2xFYVE and arrows denote TOM20+ve/PDH-ve MDVs that do not.(C) Quantification of TOM20+ve/PDH-ve MDVs that are positive for GFP-2xFYVE are shown as a % of TOM20 MDVs positive for GFP, with each data point indicating 1 cell. MDV colocalisation were quantified by eye from images captured (n = 20-22 cells per condition from 3 independent experiments).(D) SH-SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 (green), PDH E2/E3bp (red), and TOM20 (blue). Arrowheads indicate TOM20+ve/PDH-ve MDVs positive for the early endosome marker EEA1.(E) siRNA knockdown of Parkin was performed in SH-SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 (green), Tollip (red) and Cytochrome c (blue). We observed Tollip colocalisation with TOM20+ve/PDH-ve MDVs (denoted by arrowheads), which was still maintained following Parkin siRNA knockdown.(F) SH-SY5Y cells were transfected with GFP-Tollip and treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3bp (blue). (G) Quantification of GFP-Tollip/Rab7a colocalisation using Pearson's correlation.


Figure 6 ( A ) Quantitation of Tollip vesicular clusters in SH - SY5Ys treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) for 2 hours . The percentage of cells with vesicular clusters were quantified from 7 - 23 cells for each condition from 4 independent experiments . ( B ) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours . The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments . ( C ) Endogenous Tollip and LAMP1 colocalise in SH - SY5Y cells treated with AO for 2 hours . Antibodies specific to LAMP1 ( green ) and Tollip ( red ) were used . ( D - E ) SH - SY5Y cells were transfected with either wild - type GFP - Tollip ( WT ) , GFP - Tollip lacking its N - terminus ( ∆ Nterm ) , or GFP - Tollip containing a CUE domain mutation ( CUEmut ) then treated with AO for 2 hours . Cells were fixed and immunostained with antibodies specific to GFP ( green ) and LAMP1 ( red ) . ( F ) The extent of colocalisation between GFP - Tollip and LAMP1 from untreated and AO treated SH - SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson ' s correlation ( 2 - 3 cells per experiment , from 2 independent experiments ) . ( G - I ) The extent of colocalisation between GFP - Tollip and LAMP1 in Tom1 KO ( G ) , ATG5 KO ( H ) , and VPS35 KO ( I ) compared to wild - type ( WT ) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson ' s correlation ( 2 - 3 cells per experiment , from 3 independent experiments ) . Western blot analysis of lysates harvested from wild - type ( WT ) alongside either Tom1 KO ( G ) or VPS35 KO ( I ) Hela cells was performed using antibodies specific to the indicated proteins .

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Figure 6(A) Quantitation of Tollip vesicular clusters in SH-SY5Ys treated with 5 μM antimycin A/10 μM oligomycin B (AO) for 2 hours. The percentage of cells with vesicular clusters were quantified from 7-23 cells for each condition from 4 independent experiments.(B) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours. The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments.(C) Endogenous Tollip and LAMP1 colocalise in SH-SY5Y cells treated with AO for 2 hours. Antibodies specific to LAMP1 (green) and Tollip (red) were used.(D-E) SH-SY5Y cells were transfected with either wild-type GFP-Tollip (WT), GFP-Tollip lacking its N-terminus (∆Nterm), or GFP-Tollip containing a CUE domain mutation (CUEmut) then treated with AO for 2 hours. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red).(F) The extent of colocalisation between GFP-Tollip and LAMP1 from untreated and AO treated SH-SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson's correlation (2-3 cells per experiment, from 2 independent experiments). (G-I) The extent of colocalisation between GFP-Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H), and VPS35 KO (I) compared to wild-type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson's correlation (2-3 cells per experiment, from 3 independent experiments). Western blot analysis of lysates harvested from wild-type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) Hela cells was performed using antibodies specific to the indicated proteins.


Figure 7 ( A ) A subset of TOM20 + ve / PDH - ve MDVs are trafficked to the lysosome . siRNA knockdown of Tollip was conducted in SH - SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or 25 μM antimycin A ( AA ) for 2 hours . Cells were then fixed and stained with antibodies specific to LAMP1 ( green ) , PDH E2 / E3bp ( red ) and TOM20 ( blue ) . Arrows denote TOM20 + ve / PDH - ve MDVs colocalising with LAMP1 , whereas arrowheads denote MDVs that do not colocalise with LAMP1 . ( B ) From immunofluorescence images , the total number of TOM20 + ve / PDH - ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell ( n = 25 - 32 cells per condition from 3 independent experiments ) . ( C ) siRNA knockdown was performed in SH - SY5Y cells , then 18 hours prior to fixation cells were transfected with GFP - Tollip wild - type ( WT ) , ∆ Nterm , CUEmut , or R78A . Cells were fixed and immunostained for PDH E2 / E3bp ( red ) and TOM20 ( blue ) . Arrowheads denote TOM20 + ve / PDH - ve MDVs . ( D ) Quantification of TOM20 MDVs per cell was performed by eye ( n = 11 - 14 cells per mutant from 2 independent experiments ) . Cells expressing a GFP - Tollip construct were identified using the GFP channel . Surrounding cells not expressing GFP were counted as the Tollip siRNA only population .

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 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

Figure 7(A) A subset of TOM20+ve/PDH-ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH-SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM antimycin A (AA) for 2 hours. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3bp (red) and TOM20 (blue). Arrows denote TOM20+ve/PDH-ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. (B) From immunofluorescence images, the total number of TOM20+ve/PDH-ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell (n = 25-32 cells per condition from 3 independent experiments).(C) siRNA knockdown was performed in SH-SY5Y cells, then 18 hours prior to fixation cells were transfected with GFP-Tollip wild-type (WT), ∆Nterm, CUEmut, or R78A. Cells were fixed and immunostained for PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs. (D) Quantification of TOM20 MDVs per cell was performed by eye (n = 11-14 cells per mutant from 2 independent experiments). Cells expressing a GFP-Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population.