Figure 5B - D . Differentation of different CD8 + T cell populations ( B , early effector cells , EECs ( CD127lo KLRG1lo ) ; C , memory precursor effector cells ( MPECs ( CD127hi KLRG1lo ) ; D , short - lived effector cells , SLECs ( CD127lo KLRG1hi ) ) , gated on CD3 + CD8 + T cells in the presence of the indicated B cells , 2 days after a second bacterial rechallenge ( as indicated in A ) . Data information : Bars in the figure represent the mean . Error bars indicate the SD . Statistical significant differences , analyzed by ANOVA are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) . … F . Kaplan Meier survival curve of Rag1 - / - mice after adoptive transfer and bacteria challenge . Data information : Bars in the figure represent the mean . Error bars indicate the SD . Statistical significant differences , analyzed by ANOVA are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) . … H . BacB cells reduce melanoma growth . B16 F10 melanoma cells ( 4 × 105 ) were injected s . c . in the mid - right flank of C57BL / 6J ( WT ) host mice ( 4 mice / group ; treated with BacB or untreated ) . Once tumors were formed and visible ( 6 days after imlantation ) mice were treated with Listeria TRP2 BacB cells or with vehicle ( PBS ) ( three consecutive injections in three days ) . Tumor growth were monitored every 1 - 3 days . It is shown the mean and the SD of the tumor volume in each group at the indicated time points . Mice with tumors ≥ 300 mm3 were sacrified . Data information : Bars in the figure represent the mean . Error bars indicate the SD . Statistical significant differences , analyzed by ANOVA are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) . …

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 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

Figure 5B-D. Differentation of different CD8+ T cell populations (B, early effector cells, EECs (CD127lo KLRG1lo); C, memory precursor effector cells (MPECs (CD127hi KLRG1lo); D, short-lived effector cells, SLECs (CD127lo KLRG1hi)), gated on CD3+CD8+ T cells in the presence of the indicated B cells, 2 days after a second bacterial rechallenge (as indicated in A). Data information: Bars in the figure represent the mean. Error bars indicate the SD. Statistical significant differences, analyzed by ANOVA are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns). …F. Kaplan Meier survival curve of Rag1-/- mice after adoptive transfer and bacteria challenge. Data information: Bars in the figure represent the mean. Error bars indicate the SD. Statistical significant differences, analyzed by ANOVA are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns). …H. BacB cells reduce melanoma growth. B16 F10 melanoma cells (4 × 105) were injected s.c. in the mid-right flank of C57BL/6J (WT) host mice (4 mice/group; treated with BacB or untreated). Once tumors were formed and visible (6 days after imlantation) mice were treated with Listeria TRP2 BacB cells or with vehicle (PBS) (three consecutive injections in three days). Tumor growth were monitored every 1-3 days. It is shown the mean and the SD of the tumor volume in each group at the indicated time points. Mice with tumors ≥ 300 mm3 were sacrified. Data information: Bars in the figure represent the mean. Error bars indicate the SD. Statistical significant differences, analyzed by ANOVA are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns). …


Figure 1B Observed dsDNA processing patterns of Sgs1 , including an overview over consecutive bursts and pauses ( upper panel ) as well as detailed views into individual bursts containing multiple unwinding events ( lower panels ) . A typical unwinding event of Sgs1 starts with slow , gradual unwinding of the dsDNA followed by DNA rehybridization , that can be almost instant ( 76 % of events , see blue sections in lower right panel ) or contain slow rewinding sections ( 24 % of events , see orange sections in lower right panel ) . C Histograms of the observed unwinding and rewinding velocities for Sgs1 . The mean unwinding rate was 65 ± 2 bp / s ( N = 899 ) . The mean rewinding rate was 115 ± 6 bp / s ( N = 287 ) . We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections .

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 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

Figure 1B Observed dsDNA processing patterns of Sgs1, including an overview over consecutive bursts and pauses (upper panel) as well as detailed views into individual bursts containing multiple unwinding events (lower panels). A typical unwinding event of Sgs1 starts with slow, gradual unwinding of the dsDNA followed by DNA rehybridization, that can be almost instant (76% of events, see blue sections in lower right panel) or contain slow rewinding sections (24% of events, see orange sections in lower right panel).C Histograms of the observed unwinding and rewinding velocities for Sgs1. The mean unwinding rate was 65 ± 2 bp/s (N = 899). The mean rewinding rate was 115 ± 6 bp/s (N = 287). We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections.


Figure 2B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses ( upper panel ) and detailed views of single bursts ( lower panels ) . Unwinding events are always followed by slow rewinding events ( orange sections ) . C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA ( brown bars ) . The mean unwinding rate was 51 ± 3 bp / s ( N = 2139 ) . The mean rewinding rate was 66 ± 3 bp / s ( N = 2091 ) . White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1 aloneD Force - dependence of the Sgs1 unwinding ( black squares ) ( N = 2139 ) and rewinding rates ( black diamonds ) ( N = 2091 ) in the presence of RPA ( errors given as standard deviations ) . Red circles represent the force - dependent DNA opening and closure rates measured for RPA

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

Figure 2B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses (upper panel) and detailed views of single bursts (lower panels). Unwinding events are always followed by slow rewinding events (orange sections).C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA (brown bars). The mean unwinding rate was 51 ± 3 bp/s (N = 2139). The mean rewinding rate was 66 ± 3 bp/s (N = 2091). White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1 aloneD Force-dependence of the Sgs1 unwinding (black squares) (N = 2139) and rewinding rates (black diamonds) (N = 2091) in the presence of RPA (errors given as standard deviations). Red circles represent the force-dependent DNA opening and closure rates measured for RPA alone


Figure 3B Typical dsDNA processing event . The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease - dead Dna2 E675A and RPA . C Histograms of the observed unwinding and rewinding rates ( green bars ) with mean values of 69 ± 3 bp / s ( N = 365 ) and 74 ± 5 bp / s ( N = 282 ) , respectively . White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of

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

Figure 3B Typical dsDNA processing event. The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease-dead Dna2 E675A and RPA.C Histograms of the observed unwinding and rewinding rates (green bars) with mean values of 69 ± 3 bp/s (N = 365) and 74 ± 5 bp/s (N = 282), respectively. White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of RPA


Figure 4B dsDNA processing events observed for Sgs1 in the presence of Top3 - Rmi1 . Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone . C Histogram of the unwinding rate of Sgs1 in the presence of Top3 - Rmi1 ( violet bars ) with a mean rate of 86 ± 3 bp / s ( N = 1101 ) . For comparison , the distribution of unwinding rates for Sgs1 alone is depicted with white barsE dsDNA processing by Sgs1 in the presence of Top3 - Rmi1 and RPA . In contrast to the absence of RPA , DNA closure is seen as a slow rewinding . F Histograms of unwinding and rewinding rates of Sgs1 in the presence of Top3 - Rmi1 and RPA ( purple bars ) . The mean rates for unwinding and rewinding are 66 ± 2 bp / s ( N = 502 ) and 70 ± 4 bp / s ( N = 369 ) , respectively . For comparison , the velocity distributions for Sgs1 in the presence of RPA are shown as white

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 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 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

Figure 4B dsDNA processing events observed for Sgs1 in the presence of Top3-Rmi1. Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone.C Histogram of the unwinding rate of Sgs1 in the presence of Top3-Rmi1 (violet bars) with a mean rate of 86 ± 3 bp/s (N = 1101). For comparison, the distribution of unwinding rates for Sgs1 alone is depicted with white barsE dsDNA processing by Sgs1 in the presence of Top3-Rmi1 and RPA. In contrast to the absence of RPA, DNA closure is seen as a slow rewinding.F Histograms of unwinding and rewinding rates of Sgs1 in the presence of Top3-Rmi1 and RPA (purple bars). The mean rates for unwinding and rewinding are 66 ± 2 bp/s (N = 502) and 70 ± 4 bp/s (N = 369), respectively. For comparison, the velocity distributions for Sgs1 in the presence of RPA are shown as white bars


Figure 5A Mean duration of bursts ( violet bars ) ( N > 40 ) and pauses ( green bars ) ( N > 40 ) for the different enzyme combinations . Error bars represent standard error of the mean . B Mean duration of a unidirectional unwinding event ( N > 350 ) . Error bars represent standard error of the mean . C Mean processivity of unidirectional DNA unwinding ( N > 350 ) . Error bars represent standard error of the mean .

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

Figure 5A Mean duration of bursts (violet bars) (N > 40) and pauses (green bars) (N > 40) for the different enzyme combinations. Error bars represent standard error of the mean.B Mean duration of a unidirectional unwinding event (N > 350). Error bars represent standard error of the mean.C Mean processivity of unidirectional DNA unwinding (N > 350). Error bars represent standard error of the mean.


Figure 1 ( B ) Total lysates from HEK293T transfected with Flag - OFD1 were IP with anti - myosin VI antibodies ( 1295 and 1296 ) and an unrelated rabbit antibody ( anti - GST , as control ) . IB was performed with anti - Flag and anti - myosin VI antibodies . ( C ) Total lysates from HEK293T transfected with Flag - OFD1 or Flag ( as control ) were IP with anti - Flag antibody - conjugated beads . IB was performed with anti - Flag and anti - myosin VI antibodies . ( D ) Selected sections deriving from TEM analysis of A549 wild - type versus myosin VI KO cells . The cells were immunogold - labelled with anti - myosin VI antibody . The centrioles in the images are indicated with white dashed circles . Scale bar , 200 nm . Top : representation of the estimated localisation of myosin VI at the centrioles . ( F ) GST pulldown assay using myosin VI tail ( 835 - 1295 ) construct ( or GST alone as control ) and lysates from HEK293T cells transfected with the indicated Flag - OFD1 constructs ( or Flag alone , as control ) . IB was performed with anti - Flag antibody . Ponceau staining as indicated .

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 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 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

Figure 1(B) Total lysates from HEK293T transfected with Flag-OFD1 were IP with anti-myosin VI antibodies (1295 and 1296) and an unrelated rabbit antibody (anti-GST, as control). IB was performed with anti-Flag and anti-myosin VI antibodies. (C) Total lysates from HEK293T transfected with Flag-OFD1 or Flag (as control) were IP with anti-Flag antibody-conjugated beads. IB was performed with anti-Flag and anti-myosin VI antibodies.(D) Selected sections deriving from TEM analysis of A549 wild-type versus myosin VI KO cells. The cells were immunogold-labelled with anti-myosin VI antibody. The centrioles in the images are indicated with white dashed circles. Scale bar, 200 nm. Top: representation of the estimated localisation of myosin VI at the centrioles.(F) GST pulldown assay using myosin VI tail (835-1295) construct (or GST alone as control) and lysates from HEK293T cells transfected with the indicated Flag-OFD1 constructs (or Flag alone, as control). IB was performed with anti-Flag antibody. Ponceau staining as indicated.


Figure 2D ) Quantification of OFD1 intensity at the mother or daughter centrioles . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mother centrioles : Mock , n = 148 cells ; MyoVI KD , n = 199 cells , from four independent experiments . Daughter centrioles : Mock , n = 101 cells ; MyoVI KD , n = 148 cells , from three independent experiments . * * * * P < 0 , 0001 by Mann - Whitney test . ( F - G ) IF analysis of Cep164 signal . hTERT - RPE1 cells were transfected with siRNA against myosin VI and / or OFD1 . Four days after transfection , cells were treated with nocodazole ( 1 hour , 6 μg / ml ) and immunostained with anti - OFD1 , anti - centrin1 and anti - Cep164 antibodies . ( F ) Representative images . Scale bar , 1 μm . ( G ) Quantification of Cep164 intensity at the mother centrioles . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock , n = 147 cells ; OFD1 KD , n = 146 cells ; MyoVI KD , n = 150 cells ; MyoVI + OFD1 KD , n = 151 cells , from four independent experiments . * * P < 0 , 005 ; * * * * P < 0 , 0001 by Kruskal - Wallis test .

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 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

Figure 2D) Quantification of OFD1 intensity at the mother or daughter centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n=148 cells; MyoVI KD, n=199 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; MyoVI KD, n=148 cells, from three independent experiments. **** P<0,0001 by Mann-Whitney test.(F-G) IF analysis of Cep164 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. (F) Representative images. Scale bar, 1 μm. (G) Quantification of Cep164 intensity at the mother centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=147 cells; OFD1 KD, n=146 cells; MyoVI KD, n=150 cells; MyoVI + OFD1 KD, n=151 cells, from four independent experiments. ** P<0,005; **** P<0,0001 by Kruskal-Wallis test.


Figure 3 ( A ) Growth curve of hTERT - RPE1 cells transfected with myosin VI siRNA . A representative plot of three independent experiments is shown . ( B ) Growth curve of BJ - hTERT cells transfected with myosin VI siRNA . A representative plot of two independent experiments is shown . ( G ) Representative bright field images of cells treated with the indicated p53 and myosin VI siRNAs . Scale bar , 200 μm . ( I ) Quantification of OFD1 intensity at the mother or daughter centrioles . hTERT - RPE1 cells treated or not with Nutlin - 3 , were incubated with nocodazole for 1 hour ( 6 μg / ml ) and immunostained with anti - OFD1 , anti - centrin1 and anti - Cep164 antibodies . Mother centrioles were identified by the coincident staining of centrin1 and Cep164 , while daughter centrioles were centrin1 - only stained . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mother centrioles : Mock , n = 148 cells ; Nutlin - 3 , n = 169 cells , from four independent experiments . Daughter centrioles : Mock , n = 101 cells ; Nutlin - 3 , n = 122 cells , from three independent experiments . ns , not significant by Mann - Whitney test . ( J ) Quantification of Cep164 intensity at the mother centrioles in hTERT - RPE1 cells treated as in ( I ) . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock , n = 195 cells ; Nutlin - 3 , n = 196 cells , from four independent experiments . ns , not significant by Mann - Whitney test .

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 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 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 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

Figure 3(A) Growth curve of hTERT-RPE1 cells transfected with myosin VI siRNA. A representative plot of three independent experiments is shown. (B) Growth curve of BJ-hTERT cells transfected with myosin VI siRNA. A representative plot of two independent experiments is shown.(G) Representative bright field images of cells treated with the indicated p53 and myosin VI siRNAs. Scale bar, 200 μm.(I) Quantification of OFD1 intensity at the mother or daughter centrioles. hTERT-RPE1 cells treated or not with Nutlin-3, were incubated with nocodazole for 1 hour (6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mother centrioles: Mock, n=148 cells; Nutlin-3, n=169 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; Nutlin-3, n=122 cells, from three independent experiments. ns, not significant by Mann-Whitney test.(J) Quantification of Cep164 intensity at the mother centrioles in hTERT-RPE1 cells treated as in (I). Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=195 cells; Nutlin-3, n=196 cells, from four independent experiments. ns, not significant by Mann-Whitney test.


Figure 4 ( A ) Super - resolution analysis of OFD1 localisation at the centrioles . hTERT - RPE1 cells were transfected with siRNA against myosin VI , immunostained with anti - OFD1 and anti - acetylated tubulin antibodies and visualised using structured illumination microscopy ( SIM ) . Representative images are shown , scale bar , 500 nm . A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side . ( C ) dSTORM super - resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI - depleted cells . To emphasise the symmetry of the structures , the signals from all nine appendages were averaged ( bottom ) . Representative images are shown ; scale bar , 200 nm . FRAP analysis of centriole - associated GFP - OFD1 . hTERT - RPE cells stably expressing GFP - OFD1 and centrin1 - dTomato were transfected with siRNA against myosin VI . After four days , cells were treated with nocodazole ( 1 hour , 6 μg / ml ) and subjected to live - cell imaging . ( E ) A representative graph of one out of three experiments . For each time point , the fraction of recovery of GFP - OFD1 is shown . Results are expressed as means with 95 % confidence interval . n = 12 cells ( Mock ) , n = 13 cells ( MyoVI KD ) . ( F ) Quantification of the half - time of fluorescence recovery ( t1 / 2 , ) and of the mobile fraction of GFP - OFD1 . Results are expressed as mean ± SD . n = 41 cells ( Mock ) , n = 31 cells ( MyoVI KD ) , from three independent experiments . ns , not significant ; * * * P < 0 , 0005 by Unpaired T - test .

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

Figure 4(A) Super-resolution analysis of OFD1 localisation at the centrioles. hTERT-RPE1 cells were transfected with siRNA against myosin VI, immunostained with anti-OFD1 and anti-acetylated tubulin antibodies and visualised using structured illumination microscopy (SIM). Representative images are shown, scale bar, 500 nm. A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side.(C) dSTORM super-resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI-depleted cells. To emphasise the symmetry of the structures, the signals from all nine appendages were averaged (bottom). Representative images are shown; scale bar, 200 nm.FRAP analysis of centriole-associated GFP-OFD1. hTERT-RPE cells stably expressing GFP-OFD1 and centrin1-dTomato were transfected with siRNA against myosin VI. After four days, cells were treated with nocodazole (1 hour, 6 μg/ml) and subjected to live-cell imaging. (E) A representative graph of one out of three experiments. For each time point, the fraction of recovery of GFP-OFD1 is shown. Results are expressed as means with 95% confidence interval. n=12 cells (Mock), n=13 cells (MyoVI KD). (F) Quantification of the half-time of fluorescence recovery (t1/2,) and of the mobile fraction of GFP-OFD1. Results are expressed as mean ±SD. n=41 cells (Mock), n=31 cells (MyoVI KD), from three independent experiments. ns, not significant; *** P<0,0005 by Unpaired T-test.


Figure 5 ( B ) IF analysis of PCM1 signal . hTERT - RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti - PCM1 and anti - Cep135 antibodies . Upper panel , representative images , scale bar , 2 μm . Lower panel , quantification of PCM1 intensity . Results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock , n = 96 cell ; MyoVI KD , n = 98cells , from two independent experiments . * * * * P < 0 , 0001 by Mann - Whitney test . ( C ) IF analysis of PCM1 signal . hTERT - RPE1 cells were treated with Nutlin - 3 for 24 hours and immunostained with anti - PCM1 and anti - Cep135 antibodies . Panels as in B . Mock , n = 96 cells ; Nutlin - 3 , n = 100 cells , from two independent experiments . * * * * P < 0 , 0001 by Mann - Whitney test . ( D ) IF analysis of PCM1 signal . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti - PCM1 and anti - Cep164 antibodies . Panels as in B . Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole , identified with anti - Cep164 staining . Mock , n = 128 cells ; MyoVI KD , n = 114 cells from three independent experiments . ns , not significant by Mann - Whitney test .

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Figure 5(B) IF analysis of PCM1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep135 antibodies. Upper panel, representative images, scale bar, 2 μm. Lower panel, quantification of PCM1 intensity. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=96 cell; MyoVI KD, n=98cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (C) IF analysis of PCM1 signal. hTERT-RPE1 cells were treated with Nutlin-3 for 24 hours and immunostained with anti-PCM1 and anti-Cep135 antibodies. Panels as in B. Mock, n=96 cells; Nutlin-3, n=100 cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (D) IF analysis of PCM1 signal. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep164 antibodies. Panels as in B. Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole, identified with anti-Cep164 staining. Mock, n=128 cells; MyoVI KD, n=114 cells from three independent experiments. ns, not significant by Mann-Whitney test.


Figure 6 ( B ) IF analysis of OFD1 signal at the centriolar satellites upon serum starvation . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI . After four days , cells were fixed ( growing ) or serum starved for 24 hours ( SS ) . Cells were immunostained with anti - OFD1 and anti - PCM1 antibodies . The intensity of OFD1 signal in the area covered by PCM1 was quantified and normalised against the intensity of PCM1 staining in the same area . Left , representative images , scale bar , 2 μm . Right , results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . Mock _ growing , n = 150 cells ; Mock _ SS , n = 149 cells ; MyoVI KD _ growing , n = 149 cells ; MyoVI KD _ SS , n = 150 cells , from three independent experiments . ns , not significant ; * P < 0 , 05 ; * * * * P < 0 , 0001 by Kruskal - Wallis test . ( C ) IB analysis of OFD1 after serum starvation in control and myosin VI - depleted cells . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI . After four days , cells were serum starved for 24 hours ( SS ) . Lysates were analysed by IB with anti - OFD1 , anti - myosin VI , anti - p62 , and anti - LC3 antibodies . Anti - GAPDH was used as loading control . The amount of OFD1 protein was normalised against GAPDH signal and is expressed as percentage of OFD1 levels in cells grown in serum - starved conditions compared to cells grown in media containing serum . Bars represent mean ± SD . n = 5 independent experiments . * P < 0 , 05 by Kruskal - Wallis test . ( D ) IF analysis of primary cilium upon serum starvation . hTERT - RPE1 p53 KO cells were transfected with siRNA against myosin VI . After four days , cells were fixed ( growing ) or serum starved for 24 hours ( SS ) . Cells were immunostained with anti - acetylated tubulin ( to identify the cilia ) , anti - Cep135 ( to identify the centrioles ) and DAPI . Left , representative images , scale bar , 2 μm . Right , results are expressed as fold change with respect to mock average intensity . Bars represent mean ± SD . n = 3 independent experiments . 100 - 200 cells / condition were counted for each experiment . * * * P < 0 . 001 by two - way ANOVA test .

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Figure 6(B) IF analysis of OFD1 signal at the centriolar satellites upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-OFD1 and anti-PCM1 antibodies. The intensity of OFD1 signal in the area covered by PCM1 was quantified and normalised against the intensity of PCM1 staining in the same area. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock_growing, n=150 cells; Mock_SS, n=149 cells; MyoVI KD_growing, n=149 cells; MyoVI KD_SS, n=150 cells, from three independent experiments. ns, not significant; * P<0,05; **** P<0,0001 by Kruskal-Wallis test.(C) IB analysis of OFD1 after serum starvation in control and myosin VI-depleted cells. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were serum starved for 24 hours (SS). Lysates were analysed by IB with anti-OFD1, anti-myosin VI, anti-p62, and anti-LC3 antibodies. Anti-GAPDH was used as loading control. The amount of OFD1 protein was normalised against GAPDH signal and is expressed as percentage of OFD1 levels in cells grown in serum-starved conditions compared to cells grown in media containing serum. Bars represent mean ±SD. n=5 independent experiments. * P<0,05 by Kruskal-Wallis test.(D) IF analysis of primary cilium upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-acetylated tubulin (to identify the cilia), anti-Cep135 (to identify the centrioles) and DAPI. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. n=3 independent experiments. 100-200 cells/condition were counted for each experiment. ***P < 0.001 by two-way ANOVA test.


Figure 1Immunoblots of acyl - biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation - deficient mutant form of the protein with cysteines replaced with alanine residues . The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull - down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein . Immunoblots of acyl - biotin exchange purifications from Hela cells treated with either DHHC5 siRNA or control non - targeting siRNA . Immunoblots showing rescue of Golga7b palmitoylation in DHHC5 depleted cells after transfection of WT DHHC5 but not by the DHHC5 catalytic mutant . A significant reduction in the Golga7b signal is observed in the + HA condition of the pull - down when DHHC5 is depleted with siRNA . The graph displays mean + / - s . e . m . N = 4 , * = p < 0 . 05 , paired t - test . Immunoblots of WT and non - palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants ( DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine , C - terminal mutant = mutant where the 3 C - terminal palmitoylation sites of DHHC5 have been mutated to alanine residues . ) . WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C - terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C - terminus of DHHC5 is essential for the stability of this interaction .

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Figure 1Immunoblots of acyl-biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation-deficient mutant form of the protein with cysteines replaced with alanine residues. The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull-down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein.Immunoblots of acyl-biotin exchange purifications from Hela cells treated with either DHHC5 siRNA or control non-targeting siRNA.Immunoblots showing rescue of Golga7b palmitoylation in DHHC5 depleted cells after transfection of WT DHHC5 but not by the DHHC5 catalytic mutant.A significant reduction in the Golga7b signal is observed in the + HA condition of the pull-down when DHHC5 is depleted with siRNA. The graph displays mean +/- s.e.m. N=4, *=p<0.05, paired t-test.Immunoblots of WT and non-palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants (DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine, C-terminal mutant = mutant where the 3 C-terminal palmitoylation sites of DHHC5 have been mutated to alanine residues.). WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C-terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C-terminus of DHHC5 is essential for the stability of this interaction.


Figure 2Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5 . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and the catalytic mutant DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Quantification of the plasma membrane signal of DHHC5 in each of the images ( A - F ) , the graph displays mean + / - s . e . m . * * * * = p < 0 . 001 , n . s . = not significant , unpaired t - test . N = 35 , DHHC5 + WT Golga7b . N = 41 , DHHC5 + mutant Golga7b . N = 35 , DHHS5 + WT Golga7b . N = 31 , DHHS5 + Golga7b mutant . N = 29 , C - terminal mutant + WT Golga7b . N = 30 , C - terminal mutant + mutant Golga7b . Co expression of palmitoylation - deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane . The majority of the DHHC5 C - terminal palmitoylation - deficient mutant is localised to the plasma membrane in a Golga7b independent manner . All N numbers refer to the number of cells imaged and quantified , all experiments repeated 4 times . Data information : All images show ectopic protein expression only . All scale bars 10µm .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5. Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and the catalytic mutant DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Quantification of the plasma membrane signal of DHHC5 in each of the images (A-F), the graph displays mean +/- s.e.m. ****=p<0.001, n.s. = not significant, unpaired t-test. N=35, DHHC5 + WT Golga7b. N=41, DHHC5 + mutant Golga7b. N=35, DHHS5 + WT Golga7b. N=31, DHHS5 + Golga7b mutant. N=29, C-terminal mutant + WT Golga7b. N=30, C-terminal mutant + mutant Golga7b. Co expression of palmitoylation-deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane. The majority of the DHHC5 C-terminal palmitoylation-deficient mutant is localised to the plasma membrane in a Golga7b independent manner. All N numbers refer to the number of cells imaged and quantified, all experiments repeated 4 times. Data information: All images show ectopic protein expression only. All scale bars 10µm.


Figure 3Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged WT Golga7b . Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged mutant Golga7b . Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non - targeting siRNA . Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA . Quantification of plasma membrane signal of endogenous DHHC5 when co - expressed with WT or mutant Golga7b or without any transfected Golga7b . * * * * = P < 0 . 001 , t - test , endogenous + WT Golga7b n = 37 , endogenous + mutant Golga7b n = 23 , endogenous n = 42 . Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell , while WT Golga7b stabilises DHHC5 at the plasma membrane . Quantification of the plasma membrane signal of DHHC5 in ( C and D ) , * * * * = p < 0 . 001 t - test , control siRNA n = 23 , Golga7b siRNA n = 23 .

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Figure 3Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged WT Golga7b. Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged mutant Golga7b.Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non-targeting siRNA. Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA.Quantification of plasma membrane signal of endogenous DHHC5 when co-expressed with WT or mutant Golga7b or without any transfected Golga7b. ****=P<0.001, t-test, endogenous + WT Golga7b n=37, endogenous + mutant Golga7b n=23, endogenous n=42. Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell, while WT Golga7b stabilises DHHC5 at the plasma membrane.Quantification of the plasma membrane signal of DHHC5 in (C and D), ****=p<0.001 t-test, control siRNA n=23, Golga7b siRNA n=23.


Figure 4FLAG - DHHC5 immunoprecipitations were performed using lysates from Hela cells co - expressing either wild - type ( WT ) or palmitoylation deficient mutant ( m ) Golga7b . The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b , quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG - DHHC5 is expressed alone or co - expressed with either WT or mutant Golga7b . IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation . Volcano plot of DHHC5 interactors from AP - MS analysis from HeLa cells co - expressing FLAG - DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin - mediated endocytosis , cell : cell adhesion and known substrates of DHHC5 . Validation of novel DHHC5 interactors by co - IP : immunoblots of Dsg2from Hela cells co - expressing DHHC5 and either Wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of clathrin heavy chain from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of ZO - 1 protein from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * * = p < 0 . 01 ratio paired t - test , n = 3 .

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Figure 4FLAG-DHHC5 immunoprecipitations were performed using lysates from Hela cells co-expressing either wild-type (WT) or palmitoylation deficient mutant (m) Golga7b. The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b, quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG-DHHC5 is expressed alone or co-expressed with either WT or mutant Golga7b. IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation.Volcano plot of DHHC5 interactors from AP-MS analysis from HeLa cells co-expressing FLAG-DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin-mediated endocytosis, cell:cell adhesion and known substrates of DHHC5.Validation of novel DHHC5 interactors by co-IP: immunoblots of Dsg2from Hela cells co-expressing DHHC5 and either Wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of clathrin heavy chain from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of ZO-1 protein from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, **=p<0.01 ratio paired t-test, n=3.


Figure 530 minute DMSO treatment of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) . 30 minute Dynasore treatment ( 25µM ) of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) . Non - targeting control siRNA treatment ( 10nM ) of HeLa cells expressing mutant Golga7b ( HA tagged ) . Images are detecting endogenous DHHC5 and the HA tagged Golga7b AP2µ siRNA treatment ( 10nM ) of HeLa cells that were subsequently transfected with mutant Golga7b ( HA tagged ) . Images are detecting endogenous DHHC5 and the HA tagged Golga7b . Quantification of plasma membrane signal of DHHC5 in ( A and B ) . * * * * = p < 0 . 001 , unpaired t - test . N = 37 , + Dynasore . N = 28 , + DMSO . Quantification of plasma membrane signal of DHHC5 in ( C and D ) . * * * * = p < 0 . 001 , unpaired t - test . All scale bars 10μm . Biotin endocytosis assays of HeLa cells with endogenous DHHC5 , over - expression of DHHC5 alone , co - expression of DHHC5 and WT Golga7b , co - expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C - terminal mutant . Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5 . The lack of signal in the pull - down of the DHHC5 C - terminal mutant or when DHHC5 and WT Golga7b are co - expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions . The robust signal in the pull - down of endocytosed proteins from cells co - expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition . N = 3 , * = p < 0 . 05 , paired t test "

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Figure 530 minute DMSO treatment of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged). 30 minute Dynasore treatment (25µM) of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged).Non-targeting control siRNA treatment (10nM) of HeLa cells expressing mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b AP2µ siRNA treatment (10nM) of HeLa cells that were subsequently transfected with mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b.Quantification of plasma membrane signal of DHHC5 in (A and B). ****=p<0.001, unpaired t-test. N=37, +Dynasore. N=28, +DMSO.Quantification of plasma membrane signal of DHHC5 in (C and D). ****=p<0.001, unpaired t-test. All scale bars 10μm.Biotin endocytosis assays of HeLa cells with endogenous DHHC5, over-expression of DHHC5 alone, co-expression of DHHC5 and WT Golga7b, co-expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C-terminal mutant. Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5. The lack of signal in the pull-down of the DHHC5 C-terminal mutant or when DHHC5 and WT Golga7b are co-expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions. The robust signal in the pull-down of endocytosed proteins from cells co-expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition. N=3, *=p<0.05, paired t test"


Figure 6ABE purifications from HeLa cells treated with 10nM non - targeting siRNA ( nt siRNA ) or DHHC5 siRNA were immunoblotted for Dsg2 . Loss of the band in the + HA pull - down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5 . Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments . Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments . Confocal images of DHHC5 , Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments . See Appendix Figure S6 for quantification of data in panels B - D . Immunoblots and quantification of Dsg2 pulled - down in cell surface biotinylation assays in nt siRNA , DHHC5 siRNA or Golga7b siRNA treated conditions . Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input . A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA . * = Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium ( 5µM calcium ) conditions , with quantification of the intensity of the DSG2 signal in the pulldown normalised to input . There is a significant reduction in the Dsg2 signal in the + HA pull - down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition . * = p < 0 . 05 , paired t - test , N = 4 , error bars represent s . e . m . All scale bars 10μm .

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

Figure 6ABE purifications from HeLa cells treated with 10nM non-targeting siRNA (nt siRNA) or DHHC5 siRNA were immunoblotted for Dsg2. Loss of the band in the +HA pull-down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5.Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments.Confocal images of DHHC5, Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. See Appendix Figure S6 for quantification of data in panels B-D.Immunoblots and quantification of Dsg2 pulled-down in cell surface biotinylation assays in nt siRNA, DHHC5 siRNA or Golga7b siRNA treated conditions. Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input. A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA. *=Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium (5µM calcium) conditions, with quantification of the intensity of the DSG2 signal in the pulldown normalised to input. There is a significant reduction in the Dsg2 signal in the +HA pull-down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition. *=p<0.05, paired t-test, N=4, error bars represent s.e.m. All scale bars 10μm.


Figure 710x brightfield images of A431 cells treated with non - targeting siRNA at time 0 ( left ) and after 48 hours ( right ) of 5 ng / ml EGF treatment . 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . Quantification of cell scattering of A431 cells , showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed . 10x brightfield images of A431 cells treated with non - targeting siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . Quantification of cell scattering of A431 cells , showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion , was observed . Plot showing the resistance of A431 cells from a trans - epithelial electrical resistance assay treated with either DHHC5 siRNA , Golga7b siRNA or non - targeting siRNA . A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion .

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

Figure 710x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5 ng/ml EGF treatment. 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed.10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion, was observed.Plot showing the resistance of A431 cells from a trans-epithelial electrical resistance assay treated with either DHHC5 siRNA, Golga7b siRNA or non-targeting siRNA. A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion.


Figure 1 ( A , B ) Knockdown of hnRNPA3 increases poly - GA expression while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs . The control ( " x0 " ) vector lacks the G4C2 repeats but still contains the 5 ' flanking region and 3x TAG . ANOVA , Dunnett . ( C , D ) Overexpression of hnRNPA3 suppresses poly - GA expression . n = 2 experiments performed in duplicates . ANOVA , Tukey . ( E ) Increased repeat RNA upon hnRNPA3 knockdown . EGFP RNA levels are only slightly increased as compared to levels of the repeat RNA . n = 2 experiments performed in duplicates . ANOVA , Dunnett . ( F , G ) Knockdown of hnRNPA3 increases RNA foci . n = 3 replicates ; two tailed paired t - test , Scale bar = 20 μm . All graphs are shown as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Figure 1(A, B) Knockdown of hnRNPA3 increases poly-GA expression while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control ("x0") vector lacks the G4C2 repeats but still contains the 5' flanking region and 3x TAG. ANOVA, Dunnett.(C, D) Overexpression of hnRNPA3 suppresses poly-GA expression. n=2 experiments performed in duplicates. ANOVA, Tukey.(E) Increased repeat RNA upon hnRNPA3 knockdown. EGFP RNA levels are only slightly increased as compared to levels of the repeat RNA. n=2 experiments performed in duplicates. ANOVA, Dunnett.(F, G) Knockdown of hnRNPA3 increases RNA foci. n=3 replicates; two tailed paired t-test, Scale bar=20 μm. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.


Figure 2 ( A - C ) Rescue of repression of poly - GA and repeat RNA by wild type ( wt ) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD . ( B ) n = 3 experiments performed in duplicates ; ( C ) n = 2 experiments performed in duplicates . ANOVA , Tukey . ( D - F ) Rescue of repression of poly - GA and repeat RNA by hnRNPA3WT but not the M9 - NLS deletion mutant . ( E ) n = 3 experiments performed in duplicates ; ( F ) n = 4 experiments performed in duplicates . ANOVA , Tukey . All Graphs are shown as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( See also Figure EV1 ) .

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Figure 2(A-C) Rescue of repression of poly-GA and repeat RNA by wild type (wt) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD. (B) n=3 experiments performed in duplicates; (C) n= 2 experiments performed in duplicates. ANOVA, Tukey.(D-F) Rescue of repression of poly-GA and repeat RNA by hnRNPA3WT but not the M9-NLS deletion mutant. (E) n=3 experiments performed in duplicates; (F) n=4 experiments performed in duplicates. ANOVA, Tukey. All Graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (See also Figure EV1).


Figure 3 ( A ) poly - GA aggregates co - localize with p62 - positive deposits . ( B ) Redistribution of nuclear TDP - 43 to the cytoplasm in a poly - GA / poly - GR double - positive cell . Note that nuclei of poly - GA / poly - GR double positive cells are frequently disrupted . ( C ) Knockdown of hnRNPA3 increases repeat RNA expression . n = 2 experiments performed in duplicates . ANOVA , Dunnett . ( D ) Filter trap assay probed with anti - FLAG , anti - myc or anti - HA antibodies reveal abundant DPR production . ( E ) Relative expression level of poly - GA , poly - GP and poly - GR normalized to GR . n = 3 experiments , ANOVA , Tukey . ( F ) Expression of all the three DPRs is increased upon knockdown of hnRNPA3 . Signals from knockdown with siRNA # 8 were normalized to 1 . n = 3 experiments , ANOVA , Dunnett . ( G ) Co - expression of two / three different DPRs in single cells is increased upon hnRNPA3 knockdown . n = 3 replicates , ANOVA , Dunnett . All graphs are shown as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bars = 20μm . ( see also Figure EV2 and EV3 ) .

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Figure 3(A) poly-GA aggregates co-localize with p62-positive deposits.(B) Redistribution of nuclear TDP-43 to the cytoplasm in a poly-GA/poly-GR double-positive cell. Note that nuclei of poly-GA/poly-GR double positive cells are frequently disrupted.(C) Knockdown of hnRNPA3 increases repeat RNA expression. n=2 experiments performed in duplicates. ANOVA, Dunnett.(D) Filter trap assay probed with anti-FLAG, anti-myc or anti-HA antibodies reveal abundant DPR production. (E) Relative expression level of poly-GA, poly-GP and poly-GR normalized to GR. n=3 experiments, ANOVA, Tukey.(F) Expression of all the three DPRs is increased upon knockdown of hnRNPA3. Signals from knockdown with siRNA#8 were normalized to 1. n=3 experiments, ANOVA, Dunnett.(G) Co-expression of two/three different DPRs in single cells is increased upon hnRNPA3 knockdown. n=3 replicates, ANOVA, Dunnett. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Scale bars=20μm. (see also Figure EV2 and EV3).


Figure 4Rathippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA ( shA3 ) or a control shRNA ( shCtrl ) and tag RFP . Three days after transduction ( DIV3 + 3 ) , neurons were transfected with ( G4C2 ) 80HIGH ( + 0 ) and analyzed at ( DIV3 + 7 ) . ( A ) Neurons were fixed , immunostained and imaged by confocal microscopy . Double immunofluorescence for poly - GA aggregates ( green ) and tag RFP ( red ) . Nuclei were labeled with DAPI . Scale bar represent 20 μm . Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA ( shA3 ) or a control shRNA ( shCtrl ) and tag RFP . Three days after transduction ( DIV3 + 3 ) , neurons were transfected with ( G4C2 ) 80HIGH ( + 0 ) and analyzed at ( DIV3 + 7 ) . ( B ) Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA . Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA ( shA3 ) or a control shRNA ( shCtrl ) and tag RFP . Three days after transduction ( DIV3 + 3 ) , neurons were transfected with ( G4C2 ) 80HIGH ( + 0 ) and analyzed at ( DIV3 + 7 ) . ( C ) Poly - GA aggregates were detected in a filter trap assay using an anti - Flag antibody . ( D ) The amounts of poly - GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct . Means ± SD of three independent experiments are shown . * p < 0 . 05 by a Student ' s t - test . n = 3 replicates .

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Figure 4Rathippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (A) Neurons were fixed, immunostained and imaged by confocal microscopy. Double immunofluorescence for poly-GA aggregates (green) and tag RFP (red). Nuclei were labeled with DAPI. Scale bar represent 20 μm.Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (B) Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA.Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (C) Poly-GA aggregates were detected in a filter trap assay using an anti-Flag antibody. (D) The amounts of poly-GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct. Means ± SD of three independent experiments are shown. * p<0.05 by a Student's t-test. n= 3 replicates.


Figure 5 ( B ) Knockdown of hnRNPA3 with two independent siRNA results in selective depletion of hnRNPA3 in fibroblasts from 3 C9orf72 repeat carriers . Actin was used as a loading control . ( C ) Knockdown of hnRNPA3 increases RNA foci in cells derived from C9orf72 carriers . No G4C2 repeat RNA foci are detected in fibroblast without C9orf72 repeat expansions ( WT ) . Nucleoli were stained with anti nucleolin antibodies ( green ) and nuclei were stained with DAPI ( blue ) . ( D ) Quantification of the relative frequency of RNA foci positive cell ( RNA foci positivity ) ( fold change ) . n = 3 experiments for each case . ( E ) Quantification of the number of RNA foci per RNA foci - positive cell ( fold change ) . n = 3 experiments for each case . ( D , E ) The average foci number of non - treated ( N / T ) fibroblast was normalized to 1 in each case . Bars indicate mean . Individual points indicate mean values obtained from a single experiment ( 49 - 214 cells were counted in single experiment ) . Error bars indicate 95 % CI , ANOVA , Tukey - Kramer HSD , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( F ) Representative presentation of increased RNA foci in C9orf72 carriers upon lentiviral - mediated hnRNPA3 knockdown . ( G ) Quantification of the number of RNA foci ( fold change ) . 3 cases were analyzed . Single points indicate an average obtained from 29 - 30 cells per case . Color code labels values obtained in fibroblasts derived from the three individual patients . Two - tailed t - test . Mean ± SEM . Scale bar = 10 μm .

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Figure 5(B) Knockdown of hnRNPA3 with two independent siRNA results in selective depletion of hnRNPA3 in fibroblasts from 3 C9orf72 repeat carriers. Actin was used as a loading control.(C) Knockdown of hnRNPA3 increases RNA foci in cells derived from C9orf72 carriers. No G4C2 repeat RNA foci are detected in fibroblast without C9orf72 repeat expansions (WT). Nucleoli were stained with anti nucleolin antibodies (green) and nuclei were stained with DAPI (blue). (D) Quantification of the relative frequency of RNA foci positive cell (RNA foci positivity) (fold change). n=3 experiments for each case. (E) Quantification of the number of RNA foci per RNA foci-positive cell (fold change). n=3 experiments for each case. (D, E) The average foci number of non-treated (N/T) fibroblast was normalized to 1 in each case. Bars indicate mean. Individual points indicate mean values obtained from a single experiment (49-214 cells were counted in single experiment). Error bars indicate 95%CI, ANOVA, Tukey-Kramer HSD, *p<0.05, **p<0.01, ***p<0.001.(F) Representative presentation of increased RNA foci in C9orf72 carriers upon lentiviral-mediated hnRNPA3 knockdown. (G) Quantification of the number of RNA foci (fold change). 3 cases were analyzed. Single points indicate an average obtained from 29-30 cells per case. Color code labels values obtained in fibroblasts derived from the three individual patients. Two-tailed t-test. Mean ± SEM. Scale bar= 10 μm.


Figure 6 ( A ) Double immunofluorescence staining with anti - GA ( green ) and anti - hnRNPA3 ( red ) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases . In C9 mutation cases with low nuclear hnRNPA3 expression ( # 8 # 1 ) more poly - GA aggregates were observed as in cases with high nuclear hnRNPA3 ( # 7 ) . Inserts show examples of co - localization of poly - GA and hnRNPA3 aggregates . ( B ) Granular layer neurons of a C9orf72 mutation case with ( white arrows ) or without ( orange arrows ) nuclear clearance of hnRNPA3 . ( C ) poly - GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels ( divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups ) . Bar graph indicates mean value . Error bars indicate 95 % CI . Single points indicate mean from three micrographs per case . Note that the difference in GA positivity between both groups remains significant ( p = 0 . 0086 ) after removal of the highest outlier in the low nuclear A3 group . Two - tailed t - test . Scale bar = 10 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 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

Figure 6(A) Double immunofluorescence staining with anti-GA (green) and anti-hnRNPA3 (red) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases. In C9 mutation cases with low nuclear hnRNPA3 expression (#8 #1) more poly-GA aggregates were observed as in cases with high nuclear hnRNPA3 (#7). Inserts show examples of co-localization of poly-GA and hnRNPA3 aggregates.(B) Granular layer neurons of a C9orf72 mutation case with (white arrows) or without (orange arrows) nuclear clearance of hnRNPA3. (C) poly-GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels (divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups). Bar graph indicates mean value. Error bars indicate 95% CI. Single points indicate mean from three micrographs per case. Note that the difference in GA positivity between both groups remains significant (p = 0.0086) after removal of the highest outlier in the low nuclear A3 group. Two-tailed t-test. Scale bar = 10 μm.


Figure 1 ( A ) HeLa cells stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( right panel ) were transfected with either non ‐ targeting siRNA ( control siRNA ) or a pool of LC3 siRNAs ( A , B , C ) , using DharmaFect reagent . After 72 h interval , the cells were incubated for 2 h in EBSS medium in the absence or presence of 0 . 1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer . ( B ) Quantification of relative p62 and GFP ‐ GATE ‐ 16 level ( upper panel ) and degradation ( lower panel ) was performed as described in ' Materials and methods ' . Mean ± s . d . of three independent experiments is presented . ( C ) HeLa cells stably expressing GFP ‐ GATE ‐ 16 were transfected with non ‐ targeting siRNA or a pool of LC3 siRNAs and treated as in ( A ) . The cells were subjected to immunostaining with anti ‐ p62 after fixation , and analysed by confocal microscopy . The GFP intensity of three independent experiments was measured as described in ' Materials and methods ' ( right panel ) . Scale bar : 20 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.(B) Quantification of relative p62 and GFP‐GATE‐16 level (upper panel) and degradation (lower panel) was performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.(C) HeLa cells stably expressing GFP‐GATE‐16 were transfected with non‐targeting siRNA or a pool of LC3 siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.


Figure 2 ( A ) HeLa cells stably expressing GFP ‐ GATE ‐ 16 ( left panel ) or GFP ‐ LC3B ( right panel ) were transfected with either non ‐ targeting siRNA ( control siRNA ) or a pool of GABARAP siRNAs ( GABARAP , GABARAPL1 , and GATE ‐ 16 ) using DharmaFect reagent . After 72 h interval , the cells were incubated for 2 h in EBSS medium in the absence or presence of 0 . 1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer . ( B ) Quantification of relative p62 and GFP ‐ LC3B level ( upper panel ) and degradation ( lower panel ) were performed as described in ' Materials and methods ' . Mean ± s . d . of three independent experiments is presented . ( C ) HeLa cells stably expressing GFP ‐ LC3B were transfected with non ‐ targeting siRNA or a pool of GABARAP siRNAs and treated as in ( A ) . The cells were subjected to immunostaining with anti ‐ p62 after fixation , and analysed by confocal microscopy . The GFP intensity of three independent experiments was measured as described in ' Materials and methods ' ( right panel ) . Scale bar : 20 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells stably expressing GFP‐GATE‐16 (left panel) or GFP‐LC3B (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE‐16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.(B) Quantification of relative p62 and GFP‐LC3B level (upper panel) and degradation (lower panel) were performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.(C) HeLa cells stably expressing GFP‐LC3B were transfected with non‐targeting siRNA or a pool of GABARAP siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.


Figure 3 ( A ) Bulk protein degradation was tested in HeLa cells that were transfected with non ‐ targeting siRNA ( control siRNA ) , a pool of GABARAP / GATE ‐ 16 siRNAs , or a pool of LC3 siRNAs . The rate of long ‐ lived protein degradation was measured in cells incubated in either αMEM ( control ) or EBSS ( starvation ) medium , in the presence or absence of 10 mM of 3 ‐ MA , 72 h after siRNA transfection . Values expressing the protein degradation percentage are represented as the mean ± s . d . of three determinations . ( B ) HeLa cells stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( right panel ) were transfected with a pool of GABARAP / GATE ‐ 16 siRNAs or a pool of LC3 , respectively , and with non ‐ targeting siRNA . Seventy ‐ two hours after transfection , the cells were collected after 5 h of starvation and the relative level of GFP ‐ LC3B or GFP ‐ GATE ‐ 16 was measured using flow cytometry . Values represent the mean ± s . d . of three experiments .

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 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

Figure 3(A) Bulk protein degradation was tested in HeLa cells that were transfected with non‐targeting siRNA (control siRNA), a pool of GABARAP/GATE‐16 siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either αMEM (control) or EBSS (starvation) medium, in the presence or absence of 10 mM of 3‐MA, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations.(B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with a pool of GABARAP/GATE‐16 siRNAs or a pool of LC3, respectively, and with non‐targeting siRNA. Seventy‐two hours after transfection, the cells were collected after 5 h of starvation and the relative level of GFP‐LC3B or GFP‐GATE‐16 was measured using flow cytometry. Values represent the mean±s.d. of three experiments.


Figure 4 ( A ) HeLa cells stably expressing YFP ‐ Atg5 were transfected with non ‐ targeting siRNA ( control siRNA ) , a pool of LC3 siRNAs , or a pool of GABARAP / GATE ‐ 16 siRNAs using DharmaFect reagent . Seventy ‐ two hours after transfection , the cells were incubated for 2 h in EBSS medium , fixed , and immunostained with anti ‐ Atg16 antibodies . Quantification of Atg5 and Atg16 ‐ labelled puncta structures from three independent experiments is presented on the right panel . Scale bar : 20 μm . ( B ) HeLa cells stably expressing YFP ‐ Atg5 were treated as in ( A ) and monitored by live microscopy . Quantification of the lifespan of Atg5 ‐ labelled puncta structures is presented . Mean ± s . d . of three independent experiments is presented . * P0 . 05 , * * P0 . 001 . ( C ) YFP ‐ Atg5 cells were transfected with siRNA pool and starved as in ( A ) . Cryo sections of fixed cells were immunolabelled using anti ‐ GFP antibodies and analysed by TEM as described in ' Materials and methods ' . Quantification of the Atg5 ‐ labelled structures size is presented at the right panel . * P0 . 05 , * * P0 . 001 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells stably expressing YFP‐Atg5 were transfected with non‐targeting siRNA (control siRNA), a pool of LC3 siRNAs, or a pool of GABARAP/GATE‐16 siRNAs using DharmaFect reagent. Seventy‐two hours after transfection, the cells were incubated for 2 h in EBSS medium, fixed, and immunostained with anti‐Atg16 antibodies. Quantification of Atg5 and Atg16‐labelled puncta structures from three independent experiments is presented on the right panel. Scale bar: 20 μm.(B) HeLa cells stably expressing YFP‐Atg5 were treated as in (A) and monitored by live microscopy. Quantification of the lifespan of Atg5‐labelled puncta structures is presented. Mean±s.d. of three independent experiments is presented. *P0.05, **P0.001.(C) YFP‐Atg5 cells were transfected with siRNA pool and starved as in (A). Cryo sections of fixed cells were immunolabelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of the Atg5‐labelled structures size is presented at the right panel. *P0.05, **P0.001.


Figure 5 ( A ) Control HeLa cells and HeLa cells stably expressing GFP ‐ LC3B or GFP ‐ GATE ‐ 16 were starved for 2 h in EBSS medium fixed and immunostained with anti ‐ Atg16 antibodies . Quantification of Atg16 ‐ labelled puncta structures from three independent experiments is presented at the right panel . Arrowheads represent Atg ‐ 16 ‐ labelled puncta . Scale bar : 20 μm . ( B ) HeLa cells stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( middle panel ) were starved for 2 h in EBSS medium . The cells were fixed and their cryo sections were immunolabelled as in ( Figure 4B ) . Quantification of the size of GFP ‐ labelled structures ( right panel ) from three independent experiments is presented . * P0 . 05 , * * P0 . 001 .

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 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

Figure 5(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were starved for 2 h in EBSS medium fixed and immunostained with anti‐Atg16 antibodies. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Arrowheads represent Atg‐16‐labelled puncta. Scale bar: 20 μm.(B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (middle panel) were starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immunolabelled as in ( Figure 4B). Quantification of the size of GFP‐labelled structures (right panel) from three independent experiments is presented. *P0.05, **P0.001.


Figure 6 ( A , B ) Control HeLa cells and HeLa cells stably expressing GFP ‐ LC3B ( A ) or GFP ‐ GATE ‐ 16 ( B ) were transfected with the reciprocal siRNA pools using DharmaFect reagent . After 72 h interval , the cells were incubated for 2 h in EBSS , subjected to immunostaining with anti ‐ Atg16 after fixation , and analysed by confocal microscopy . Quantification of cells containing Atg16 structures larger than 2 μm ( A ) or Atg16 ‐ labelled puncta structures ( B ) from three independent experiments in comparison to control and to G to A mutants ( D ) is presented at the lower panel . Scale bar : 20 μm . ( C ) HeLa cells stably expressing silent GFP ‐ LC3B or GFP ‐ GATE ‐ 16 were transfected with their siRNA pools using DharmaFect reagent . After 72 ‐ h interval , the cells were incubated for 2 h in EBSS medium and subjected to immunostaining with anti ‐ Atg16 and anti ‐ p62 antibodies followed by fixation . The cells were analysed by confocal microscopy . Arrows represent cells , which do not express GFP proteins whereas arrowheads represent cells , which express GFP ‐ proteins . Quantification of Atg16 ‐ labelled puncta structures from three independent experiments is presented at the right panel . Scale bar : 20 μm . ( D ) HeLa cells stably expressing GFP ‐ LC3BG120A or GFP ‐ GATE ‐ 16G116Awere transfected with either pool of GABARAP / GATE ‐ 16 siRNAs or LC3 siRNAs , respectively , and a non ‐ targeting siRNA . The cells were starved for 2 h , fixed , and immunostained with anti ‐ Atg16 and anti ‐ p62 antibodies . Quantification is presented in ( A , B ) at the lower panels . Scale bar : 20 μm . ( E ) HeLa cells stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( right panel ) were transfected with the reciprocal siRNA pool or with non ‐ targeting siRNA and starved for 2 h in EBSS medium . The cells were fixed and their cryo sections were immuno ‐ labelled using anti ‐ GFP antibodies and analysed by TEM as described in ' Materials and methods ' . Quantification of cells containing Atg16 structures larger than 2 μm ( left panel ) or the size of Atg16 ‐ labelled structures ( right panel ) is presented .

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 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 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 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

Figure 6(A, B) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B (A) or GFP‐GATE‐16 (B) were transfected with the reciprocal siRNA pools using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS, subjected to immunostaining with anti‐Atg16 after fixation, and analysed by confocal microscopy. Quantification of cells containing Atg16 structures larger than 2 μm (A) or Atg16‐labelled puncta structures (B) from three independent experiments in comparison to control and to G to A mutants (D) is presented at the lower panel. Scale bar: 20 μm.(C) HeLa cells stably expressing silent GFP‐LC3B or GFP‐GATE‐16 were transfected with their siRNA pools using DharmaFect reagent. After 72‐h interval, the cells were incubated for 2 h in EBSS medium and subjected to immunostaining with anti‐Atg16 and anti‐p62 antibodies followed by fixation. The cells were analysed by confocal microscopy. Arrows represent cells, which do not express GFP proteins whereas arrowheads represent cells, which express GFP‐proteins. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Scale bar: 20 μm.(D) HeLa cells stably expressing GFP‐LC3BG120A or GFP‐GATE‐16G116Awere transfected with either pool of GABARAP/GATE‐16 siRNAs or LC3 siRNAs, respectively, and a non‐targeting siRNA. The cells were starved for 2 h, fixed, and immunostained with anti‐Atg16 and anti‐p62 antibodies. Quantification is presented in (A, B) at the lower panels. Scale bar: 20 μm.(E) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with the reciprocal siRNA pool or with non‐targeting siRNA and starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immuno‐labelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of cells containing Atg16 structures larger than 2 μm (left panel) or the size of Atg16‐labelled structures (right panel) is presented.


Figure 7 ( A ) Control HeLa cells and HeLa cells stably expressing GFP ‐ LC3B or GFP ‐ GATE ‐ 16 were transfected with HsAtg4AC77A ‐ Myc ‐ His6 . After 48 h interval , the cells were incubated for 2 h in EBSS , and subjected to immunostaining with anti ‐ Atg16 and anti ‐ myc followed by confocal microscopy analysis . Quantification of cells containing Atg16 structures larger than 2 μm from three independent experiments is presented at the right panel . Scale bar : 20 μm . ( B ) HeLa cells were transfected with HsAtg4AC77A ‐ Myc ‐ His6 or empty vector ( mock ) . After a 48 h interval , the cells were incubated for 2 h in EBSS , fixed , and their ultrathin sections were analysed by TEM as described in ' Materials and methods ' . * P0 . 05 .

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 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

Figure 7(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were transfected with HsAtg4AC77A‐Myc‐His6. After 48 h interval, the cells were incubated for 2 h in EBSS, and subjected to immunostaining with anti‐Atg16 and anti‐myc followed by confocal microscopy analysis. Quantification of cells containing Atg16 structures larger than 2 μm from three independent experiments is presented at the right panel. Scale bar: 20 μm.(B) HeLa cells were transfected with HsAtg4AC77A‐Myc‐His6 or empty vector (mock). After a 48 h interval, the cells were incubated for 2 h in EBSS, fixed, and their ultrathin sections were analysed by TEM as described in 'Materials and methods'. *P0.05.