MEF2 - lacZ reporter mice ( BALB / c - background , 6 - 12 old ) were treated with 7 mg / kg lipopolysaccharide from Escherichia coli ( O111 : B4 ) or saline intraperitoneally were sacrificed after 24 hours D Induction of PKD , HDAC5 PAK2 in myocardial inflammations . Immunoblots were performed on extracts of hearts of saline - LPS - treated mice . Representative blots are shown in left panel , is in right panel Data information : values are mean ± s . e . m exact n values are shown in bottom of bar graphs . * , P < 0 . 05 , Welch ' s two - unpaired t - was used for statistical , n . s . = not significant P values ) ; phospho - PKD - Ser - 744 / Ser - 748 , P = 0 . 0049 ( D ) ; phospho - PKD - Ser - 916 , P = 0 . 2684 ( D ) ; phospho - HDAC5 - Ser - 498 ( D ) , P = 0 . 0019 ; phospho - HDAC5 - Ser - 259 , P = 0 . 0003 ( D ) ; phospho - PAK2 - Ser - 192 / Ser - 197 , P = 0 . 1962 ( D ) ; phospho - PAK2 - Thr - 402 , P = 0 . 0446 ( D ) . exact n P values can be also found in Source Data excel file for Figure

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MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD, HDAC5 and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, the quantification is in the right panel Data information: values are mean±s.e.m The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values ); phospho-PKD-Ser-744/Ser-748, P=0.0049 (D); phospho-PKD-Ser-916, P=0.2684 (D); phospho-HDAC5-Ser-498 (D), P=0.0019; phospho-HDAC5-Ser-259, P=0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P=0.1962 (D); phospho-PAK2-Thr-402, P=0.0446 (D). The exact n and P values can be also found in the Source Data excel file for Figure 7


A Isothermal calorimetry ( ITC ) assays using ADP - ribose ligand purified macrodomains of human macroH2A1 . 1 macroH2A2 .

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A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.


B In vitro PARP1 activity in presence of increasing concentrations ( 10 , 25 50 μM ) of purified macrodomains of human macroH2A1 . 1 , macroH2A1 . 1 G224E mutant macroH2A2 . PARP1 activity is assessed by its auto - PARylation detected by an anti - PAR immunoblot . naphtol blue shows increasing amounts of purified macrodomain added to each reaction . A representative blot of one of three independent experiments is shown .

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B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.


A diameter ( d ) of signaling of H2B tagged with photo - activatable GFP ( PAGFP - H2B ) is used to quantify expansion across 120 seconds after induction of damage using intercalating dye local irradiation with a .

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A The diameter (d) of the fluorescence signaling of H2B tagged with photo-activatable GFP (PAGFP-H2B) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.


B expansion in absence presence of intercalating dye ( - + damage ) pretreatment of 1h with 1μM of Olaparib PARP inhibitor . Boxplots represent single measurement of expansion at 120 sec . post damage from 3 biological replicates with n > 30 each . box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values ( * , p < 0 . 05 using unpaired , two - Student ' s T - ) .

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B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).


C Quantified expansion in not transfected ( control ) transfected with each mCherry - tagged histone ( as indicated ) . Data is represented as in B ( n > 30 , 3 bio . repl . ; * , p < 0 . 05 compared to mCherry - H2A - expressing using unpaired , two - Student ' s T - ) .

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C Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry-tagged histone (as indicated). Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 compared to mCherry-H2A-expressing cells using unpaired, two-tailed Student's T-test).


D expansion in Hela containing a doxocyclin ( doxo . ) - inducible macroH2A1 . 1 transgene . expansion was performed 48 hours after induction expression was controlled by anti - macroH2A1 . 1 - immunoblotting . Data is represented as in B ( n > 30 , 3 bio . repl . ; * , p < 0 . 05 using unpaired , two - Student ' s T - ) .

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D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).


Quantified expansion in transfected with indicated histone constructs . Boxplots represent single measurement of expansion at 120 sec . post damage ( n > 30 , 3 bio . repl . ) . box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values . ( * , p < 0 . 05 using unpaired two - Student ' s T - comparing to H2A ; n . s . , not significant ) .

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Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).


A Western blot showing expression of macroH2A in control HepG2 , knock - down for macroH2A1 macroH2A2 ( DKD ) DKD with ectopic expression of indicated YFP - tagged constructs . H3 is used as a loading control .

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A Western blot showing the expression of macroH2A proteins in control HepG2 cells, knock-down for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP-tagged constructs. H3 is used as a loading control.


B H3K9me3 immunostaining in indicated HepG2 lines with counterstaining in DAPI . indicated areas are zoomed - in for a better observation of H3K9me3 signal profile . are maximum Z - projections of confocal stacks .

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B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.


of box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values . ( * , p < 0 . 05 using unpaired two - Student ' s T - ; n . s . , not significant ) .

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Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


of H3K9me3 foci per nucleus , box limits correspond to 25th 75th percentiles , bold indicates median cross indicates average values . ( * , p < 0 . 05 using unpaired two - Student ' s T - ; n . s . , not significant ) .

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Quantification of H3K9me3 foci per nucleus, The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


Wild - type , apg12 - 1 mutant Δapg12 were cultured in nitrogen - starvation medium containing 1 mM PMSF . After incubation for 6 h , were observed under a phase - contrast microscope . Arrows indicate autophagic bodies .

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Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.


c , Wild - type ( squares ) Δapg12 ( circles ) were cultured in nitrogen - starvation medium their viability was determined by phloxine B .

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c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.


d , of autophagic activity of wild - type Δapg12 by alkaline phosphatase ( ALP ) before ( black bars ) after ( bars ) nitrogen starvation for 4 h . Error bars indicate s . d . of three independent experiments .

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d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.


a , b , Lysates from Δapg12 carrying only vector or 3 × HA - APG12 ( a ) , Δapg5 , apg7 - 1 , apg10 - 1 Δapg1 carrying 3 × HA - Apg12 plasmid ( b ) were immunoblotted using anti - HA antibody . positions of 3 × HA - Apg12 larger product ( asterisks ) are indicated .

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a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated.


c , Immunoblot of wild - type Δapg12 harbouring HA - APG5 plasmid .

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c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid.


d , Δapg5 Δapg12 were co - transformed with Myc - APG12 HA - APG5 . Their lysates were immunoprecipitated with anti - Myc or anti - HA antibodies detected by immunoblotting using anti - Myc antibody . position of crossreacting IgG heavy chain is

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d, Δapg5 Δapg12 cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicated


b , Δapg12 were transformed with mutant plasmids their lysates were immunoblotted with anti - HA antibody .

b, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody.


c , Autophagic activity was measured as described for Fig . 1d .

c, Autophagic activity was measured as described for Fig. 1d.


d , Transport of pro - API to vacuole was examined by immunoblotting with anti - API antiserum . positions of pro - API mature API are indicated .

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d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of pro-API and mature API are indicated.


Δapg5 were transformed with vector alone , wild - type APG5 or APG5K149R , then immunoblotted with anti - HA ( b )

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted with anti-HA (b)


Autophagic activity was determined by alkaline phosphatase ( c ) .

Autophagic activity was determined by alkaline phosphatase assay (c).


Δapg5 were transformed with vector alone , wild - type APG5 or APG5K149R , then immunoblotted anti - API ( d ) .

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted anti-API (d).


c , In vitro conjugation of Apg5 Apg12 . A lysate of Δapg12 carrying HA - APG5 ( lane 1 ) was incubated with an equal amount of lysate from Δapg5 carrying HA - APG12 ( 2 µ plasmid ) ( lane 2 ) at 30 ° C with ( lane 4 - 6 ) or without ( lane 7 ) 5 mM ATP . Samples were mixed with SDS - sample buffer at times indicated .

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c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.


Spheroplasts were generated from expressing either HA - Apg12 or HA - Apg5 . Their lysates were mixed layered on top of a 10 - step ( 18 - 54 % w / w ) sucrose gradient , centrifuged at 174 , 000g for 2 . 5 h ( ref . 29 ) . Fifteen fractions were collected positions of free Apg5 , free Apg12 Apg5 / Apg12 conjugate were examined by western blotting . peak fractions of alcohol dehydrogenase ( ADH ) ( cytosol ) , ALP ( vacuole ) , Kex2 ( Golgi ) Sec12 ( endoplasmic reticulum ) are indicated by arrows .

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Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.


A ) We identified previously unknown telomere regulators in fission yeast using haploid S . pombe whole - genome gene deletion library including ssu72 + ( SPAC3G9 . 04 ) . B ) Telomere of wt , ssu72Δ ssu72 - C13S ( point mutation at phosphatase active site ) strains were measured by Southern blot in ApaI digested genomic using a telomeric probe .

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A) We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04). B) Telomere length of wt, ssu72Δ and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe.


C ) Ssu72 recruitment to telomeres is cycle regulated . Ssu72 was myc - tagged at 3 ' end ChIP was carried out in cycle synchronized populations of cdc25ts strains . Septa formation was used proxy for S - phase . n ≥ 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to ssu72 + control samples . Error bars represent standard error of mean ( ) .

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C) Ssu72 recruitment to telomeres is cell cycle regulated. Ssu72 was myc-tagged at the 3' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains. Septa formation was used proxy for S-phase. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).


D ) Telomere of ssu72Δ is dependent on telomerase . Diploid strains with appropriate genotypes were sporulated trt1 ∆ ssu72 ∆ double mutants were streaked for multiple passages ( triangle indicates increased number of generations ) .

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D) Telomere length of ssu72Δ is dependent on telomerase. Diploid strains with the appropriate genotypes were sporulated and trt1∆ ssu72∆ double mutants were streaked for multiple passages (triangle indicates increased number of generations).


E ) Telomerase is recruited to telomeres in absence of Ssu72 . ChIP for Trt1 - myc in wt ssu72 ∆ was performed as described in material methods using a non - tagged as a control . n ≥ 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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E) Telomerase is recruited to telomeres in the absence of Ssu72. ChIP analysis for Trt1-myc in wt and ssu72∆ was performed as described in material and methods using a non-tagged strain as a control. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


F ) Telomerase activator Ccq1 is hyperphosphorylated in ssu72 ∆ . rap1 ∆ were used as positive control for Ccq1 phosphorylation status . Western blots were performed using Ccq1 - flag tagged strains .

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F) Telomerase activator Ccq1 is hyperphosphorylated in ssu72∆ cells. rap1∆ cells were used as positive control for Ccq1 phosphorylation status. Western blots were performed using Ccq1-flag tagged strains.


A ) ssu72Δ telomeres present longer G - rich overhangs than wt rif1 ∆ . In - gel hybridization in native denaturing conditions was labelled with a radiolabelled C - rich telomere probe quantified for ssDNA at telomeres . n = 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

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A) ssu72Δ telomeres present longer G-rich overhangs than wt and rif1∆. In-gel hybridization in native and denaturing conditions was labelled with a radiolabelled C-rich telomere probe and quantified for ssDNA at the telomeres. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


B ) RPA ( Rad11 - GFP ) is enriched at ssu72 ∆ telomeres . Colocalization of Rad11 - GFP with Taz1 - mCherry , used as a telomere marker was performed in wt ssu72 ∆ ; n = 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) . More than 1000 were analyzed for each

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B) RPA (Rad11-GFP) is enriched at ssu72∆ telomeres. Colocalization of Rad11-GFP with Taz1-mCherry, used as a telomere marker was performed in wt and ssu72∆ cells; n =3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). More than 1000 cells were analyzed for each genotype


C ) ssu72 + controls telomere independently of rif1 + . Epistasis of telomere of ssu72 ∆ ssu72 - C13S ( catalytically inactive mutant ) with rif1 ∆ was performed by Southern blotting of ApaI digested using a telomeric probe . D ) ssu72 + stn1 + regulate telomere in same genetic pathway . Epistasis of ssu72 ∆ stn1 - 75 performed by Southern blotting of ApaI digested using a telomeric probe . Two independently generated ssu72 ∆ stn1 - 75 double mutants are shown .

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C) ssu72+ controls telomere length independently of rif1+. Epistasis analysis of telomere length of ssu72∆ and ssu72-C13S (catalytically inactive mutant) with rif1∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe. D) ssu72+ and stn1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and stn1-75 performed by Southern blotting of ApaI digested DNA using a telomeric probe. Two independently generated ssu72∆ stn1-75 double mutants are shown.


A ) Ssu72 is required for telomere recruitment of Stn1 in late S phase . ChIP of stn1 - myc in wt ssu72 ∆ was performed in synchronized cdc25ts . n ≥ 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to ssu72 + control samples . Error bars represent standard error of mean ( ) .

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A) Ssu72 is required for telomere recruitment of Stn1 in late S phase. ChIP analysis of stn1-myc in wt and ssu72∆ cells was performed in synchronized cdc25ts cells. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).


B ) 2D - gel of NsiI telomeric fragments of wt ssu72 ∆ strains . Smart ladder from Eurogentec was used for measurement .

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B) 2D-gel analysis of NsiI telomeric fragments of wt and ssu72∆ strains. Smart ladder from Eurogentec was used for DNA size measurement.


C ) Serine 74 substitution to a phosphomimetic aspartate acid ( stn1 - S74D ) is sufficient to confer ssu72 ∆ telomere defects . Telomere epistasis of ssu72 ∆ stn1 - S74D mutants were performed by Southern blotting of ApaI digested genomic using a telomeric probe .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

C) Serine 74 substitution to a phosphomimetic aspartate amino acid (stn1-S74D) is sufficient to confer ssu72∆ telomere defects. Telomere length epistasis analysis of ssu72∆ and stn1-S74D mutants were performed by Southern blotting of ApaI digested genomic DNA using a telomeric probe.


E ) Similar to ssu72 ∆ mutants , stn1 - S74D is defective in telomere recruitment . ChIP of stn1 - myc stn1 - S74D - myc using a non - tagged as a control . n = 3 ; * p ≤ 0 . 05 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Similar to ssu72∆ mutants, stn1-S74D is defective in telomere recruitment. ChIP analysis of stn1-myc and stn1-S74D-myc using a non-tagged strain as a control. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


A ) ssu72 + pol1 + regulate telomere in same genetic pathway . Epistasis of ssu72 ∆ pol1 - 13 mutants was performed by Southern blotting of ApaI digested using a telomeric probe .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

A) ssu72+ and pol1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and pol1-13 mutants was performed by Southern blotting of ApaI digested DNA using a telomeric probe.


B ) Stn1 - Pol1 interaction requires Ssu72 phosphatase . Immunoprecipitation experiments of Pol1 - Flag with Stn1 - Myc was preformed both in wt ssu72 ∆ mutants . C ) Immunoprecipitation of Ten1 - Flag with Stn1 - Myc in either wt or ssu72 ∆ was performed as control for Stn1 - Ten1 integrity .

O O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O

B) Stn1-Pol1 interaction requires Ssu72 phosphatase. Immunoprecipitation experiments of Pol1-Flag with Stn1-Myc was preformed both in wt and ssu72∆ mutants. C) Immunoprecipitation of Ten1-Flag with Stn1-Myc in either wt or ssu72∆ was performed as control for Stn1-Ten1 complex integrity.


D ) Overexpression of polymerase alpha partially rescues telomere defect of ssu72Δ mutants . Multi - copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O

D) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants. Multi-copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells.


A ) Telomere elongation of SSU72 down - regulated is telomerase dependent . Telomere Southern blot was carried out with HT1080 infected for 4 with lentiviral particles carrying two independent shRNAs against SSU72 ( CDS UTR regions ) Luciferase ( Luc ) as control for shRNA . Knockdown efficiencies were determined by RT - qPCR using specific primers against hSSU72 .

O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O 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-MEASURED_VAR O

A) Telomere elongation of SSU72 down-regulated cells is telomerase dependent. Telomere Southern blot was carried out with HT1080 cells infected for 4 weeks with lentiviral particles carrying two independent shRNAs against SSU72 (CDS and UTR regions) and Luciferase (Luc) as control for shRNA. Knockdown efficiencies were determined by RT-qPCR using specific primers against hSSU72.


B ) Telomere elongation of SSU72 down - regulated is epistatic with STN1 downregulation . TRF of HT1080 infected with appropriate retroviral or lentiviral particles carrying either GFP shRNA , SSU72 shRNA ( against UTR ) , STN1 shRNA or double SSU72 shRNA / STN1 shRNA . were grown for 4 was isolated to carry out Southern Blot .

O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

B) Telomere elongation of SSU72 down-regulated cells is epistatic with STN1 downregulation. TRF analysis of HT1080 cells infected with the appropriate retroviral or lentiviral particles carrying either GFP shRNA, SSU72 shRNA (against UTR), STN1 shRNA or double SSU72 shRNA/STN1 shRNA. Cells were grown for 4 weeks and DNA was isolated to carry out Southern Blot analysis.


C ) hSSU72 down - regulation results in multi - telomeric signals ( MTS ) that are dependent on replication . Visualization of mitotic spreads of HT1080 hSSU72 shRNA treated with aphidicolin colcemid . Telomere FISH was carried out using a PNA - telomere probe . MTS are represented by arrows . of MTS per metaphase : n = 4 ; * * p ≤ 0 . 01 * * * * p ≤ 0 . 0001 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) hSSU72 down-regulation results in multi-telomeric signals (MTS) that are dependent on DNA replication. Visualization of mitotic spreads of HT1080 hSSU72 shRNA cells treated with aphidicolin and colcemid. Telomere FISH was carried out using a PNA-telomere probe. MTS are represented by arrows. Quantification of MTS per metaphase: n=4; **p ≤0.01 ****p ≤0.0001 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


A ) hSSU72 downregulation results in telomere damage foci ( TIF ) . with indicated shRNAs were fixed IF - FISH was performed using a 53BP1 antibody a PNA - telomere probe . ( Right ) of with more than 5 telomeric 53BP1 foci : n = 3 ; * * p ≤ 0 . 01 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) hSSU72 downregulation results in telomere DNA damage foci (TIF). Cells with indicated shRNAs were fixed and IF-FISH analysis was performed using a 53BP1 antibody and a PNA-telomere probe. (Right) Quantification of cells with more than 5 telomeric 53BP1 foci: n=3; **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


B ) hSSU72 is required for efficient loading of hSTN1 at human telomeres . ChIP was preformed using anti - FLAG antibodies Southern blotting was performed using a human telomeric probe . of 3 independent ChIP experiments * p ≤ 0 . 05 * * p ≤ 0 . 01 on a two - Student ' s t - to control sample . Error bars represent standard error of mean ( ) .

O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) hSSU72 is required for efficient loading of hSTN1 at human telomeres. ChIP was preformed using anti-FLAG antibodies and Southern blotting was performed using a human telomeric probe. Quantification of 3 independent ChIP experiments *p ≤0.05 **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).


A ) Volcano plot of proteome . Plotted is negative log10 of p - value ( y - axis ) versus log2 LFQ of SPPL2c overexpressing control ( x - axis ) of a given identified . A permutation FDR correction for multiple hypotheses was applied ( p = 0 . 05 ; s0 = 0 . 1 ; dashed ) . Open grey circles indicate not - significantly altered in SPPL2c expressing , red circles significantly altered after FDR correction . Filled red circles indicate type IV filled blue circles with red blue names type II altered in SPPL2c expressing , n = 6 biological repl . per .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O O O

A) Volcano plot of proteome analysis. Plotted is the negative log10 of the p-value (y-axis) versus the log2 LFQ ratio of SPPL2c overexpressing and control cells (x-axis) of a given identified protein. A permutation based FDR correction for multiple hypotheses was applied (p = 0.05; s0 = 0.1; dashed line). Open grey circles indicate proteins not-significantly altered in SPPL2c expressing cells, red circles significantly altered proteins after FDR correction. Filled red circles indicate type IV membrane proteins and filled blue circles with red line and blue names type II proteins altered in SPPL2c expressing cells, n=6 biological repl. per cell line.


C ) Western Blot of selected type IV SPPL2c candidate substrates . Endogenous of VAPA , VAPB , VAMP8 , Syntaxin 8 , 18 ( Stx8 & Stx18 ) Membrin ( encoded by gene Golgi SNAP Receptor Member 2 , GOSR2 ) were analyzed in HEK293 ectopically expressing wild type SPPL2c ( SPPL2c WT ) or a catalytically inactive SPPL2c ( SPPL2c D / A ) . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as controls . VAPA , VAPB , VAMP8 , Stx8 Membrin were decreased in SPPL2c expressing but not in control or SPPL2c D / A expressing . For Stx18 a cleavage product was detected in SPPL2c WT expressing . Expression of SPPL2c are depicted Calnexin serves as loading control .

O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O

C) Western Blot of selected type IV SPPL2c candidate substrates. Endogenous protein levels of VAPA, VAPB, VAMP8, Syntaxin 8, 18 (Stx8 & Stx18) and Membrin (encoded by the gene Golgi SNAP Receptor Complex Member 2, GOSR2) were analyzed in HEK293 cells ectopically expressing wild type SPPL2c (SPPL2c WT) or a catalytically inactive SPPL2c (SPPL2c D/A). Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. VAPA, VAPB, VAMP8, Stx8 and Membrin protein levels were decreased in SPPL2c expressing cells but not in control or SPPL2c D/A expressing cells. For Stx18 a cleavage product was detected in SPPL2c WT expressing cells. Expression levels of SPPL2c are depicted and Calnexin serves as loading control.


A ) SPPL2c overexpression causes a decrease on SPPL3 . Endogenous of SPPL3 were assessed in ectopically expressing wild type SPPL2c ( SPPL2c WT ) or catalytically inactive SPPL2c ( SPPL2c D / A ) by Western Blot . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as controls . Note that SPPL3 expression is reduced in expressing SPPL2c wt . Data information : Expression of ectopically expressed SPPL proteases are shown Calnexin serves as loading

O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O

A) SPPL2c overexpression causes a decrease on SPPL3 levels. Endogenous levels of SPPL3 were assessed in cells ectopically expressing wild type SPPL2c (SPPL2c WT) or catalytically inactive SPPL2c (SPPL2c D/A) by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. Note that SPPL3 expression is reduced in cells expressing SPPL2c wt. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control


B ) SPPL2c impairs secretion maturation of glycan - modifying enzymes . of endogenous EXTL3 , B4GALT1 , OGFOD3 GnT - V were monitored in lysates of treated with control siRNA ( siCtr ) or with siRNA targeting SPPL3 ( siSPPL3 ) in ectopically expressing either SPPL2c or SPPL3 by Western Blot . In addition , secreted forms ( sEXTL3 , sB4GALT1 , sOGFOD3 , sGnTV , ) were analyzed in corresponding conditioned media using Western Blot . Note that SPPL2c reduces secretion of glycosyltransferases similar to SPPL3 knock down , but additionally induces a decrease of mature glycosyltransferases in lysate similar to ectopic expression of SPPL3 . Data information : Expression of ectopically expressed SPPL proteases are shown Calnexin serves as loading

O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O

B) SPPL2c impairs secretion and maturation of glycan-modifying enzymes. Protein levels of endogenous EXTL3, B4GALT1, OGFOD3 and GnT-V were monitored in lysates of cells treated with control siRNA (siCtr) or with siRNA targeting SPPL3 (siSPPL3) and in cells ectopically expressing either SPPL2c or SPPL3 by Western Blot. In addition, the secreted forms (sEXTL3, sB4GALT1, sOGFOD3, sGnTV,) were analyzed in the corresponding conditioned media using Western Blot. Note that SPPL2c reduces secretion of the glycosyltransferases similar to SPPL3 knock down, but additionally induces a decrease of the mature glycosyltransferases in the cell lysate similar to ectopic expression of SPPL3. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control


C ) Processing of ectopically expressed GnT - V with an N - terminal Flag - C - terminal V5 - tag ( Flag - GnTV - V5 ) was analyzed in lysates conditioned media ( sup . ) of either SPPL2c wt or SPPL2c D / A expressing by Western Blot . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as controls . In addition , maturation of glycoproteins Nicastrin ( NCT ) , Lamp2 SPPL2a was monitored by detection of respective mature ( mat ) immature ( im ) version . Note that secretion of GnT - V is impaired in SPPL2c wt expressing immature glycoproteins accumulate . Data information : Expression of ectopically expressed SPPL proteases are shown Calnexin serves as loading

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

C) Processing of ectopically expressed GnT-V with an N-terminal Flag- and C-terminal V5-tag (Flag-GnTV-V5) was analyzed in lysates and conditioned media (sup.) of either SPPL2c wt or SPPL2c D/A expressing cells by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. In addition, maturation of the glycoproteins Nicastrin (NCT), Lamp2 and SPPL2a was monitored by detection of the respective mature (mat) and immature (im) version. Note that secretion of GnT-V is impaired in SPPL2c wt expressing cells and immature glycoproteins accumulate. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control


D ) SPPL2c induces mislocalization of GnT - V . Ectopically expressed GnT - V was visualized by immunofluorescence using anti - V5 antibody either in control ( Ctr ) or in co - expressing either SPPL2c wt or SPPL2c D / A . ER was stained using anti - BiP antibody Golgi with anti - TGN46 antibody . While in control GnT - V mainly localizes to Golgi , its localization shifts to more ER - like structures in SPPL2c expressing . Scale bar 5 µm .

O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

D) SPPL2c induces mislocalization of GnT-V. Ectopically expressed GnT-V was visualized by immunofluorescence staining using the anti-V5 antibody either in control cells (Ctr) or in cells co-expressing either SPPL2c wt or SPPL2c D/A. The ER was stained using the anti-BiP antibody and the Golgi with the anti-TGN46 antibody. While in control cells GnT-V mainly localizes to the Golgi, its localization shifts to more ER-like structures in SPPL2c expressing cells. Scale bar 5 µm.


of validated SPPL2c substrates VAPA ( A ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O

Protein levels of the validated SPPL2c substrates VAPA (A) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates VAPB ( B ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates VAPB (B) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates VAMP8 ( C ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates VAMP8 (C) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates Syntaxin 8 ( Stx8 ) ( D ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O

Protein levels of the validated SPPL2c substrates Syntaxin 8 (Stx8) (D) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates Syntaxin 18 ( Stx18 ) ( E ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates Syntaxin 18 (Stx18) (E) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


of validated SPPL2c substrates Membrin ( F ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control overexpression of SPPL2c WT was used as a positive control . Non - transfected ( - ) or stably transfected but non - induced SPPL2c wt ( n / i ) served as additional controls for antibody specificity . Processing maturation of ectopically expressed GnT - V maturation of endogenous SPPL2a were monitored by Western Blotting . None of substrates ' knock down fully recapitulated effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing . Calnexin served as loading control ectopic expression of SPPL2c WT SPPL2c D / A was confirmed using a SPPL2c specific antibody .

Protein levels of the validated SPPL2c substrates Membrin (F) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


B ) Processing of endogenous Syntaxin 5 ( Stx5 ) Syntaxin 6 ( Stx6 ) by SPPL2c wt by Western Blot . Non - transfected ( - ) , stably transfected but non - induced SPPL2c wt ( n / i ) induced stably expressing SPPL2c D / A served as controls . Results are derived from one experiment , thus expression ( SPPL2c ) loading ( Calnexin ) control are same . Note , that Syntaxin 5 is by SPPL2c while Syntaxin 6 is not .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O

B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not.


C ) Syntaxin 5 were down regulated processing maturation of ectopically expressed GnT - V , as well maturation of endogenous glycoproteins Nicastrin ( NCT ) SPPL2a was analyzed in Western Blot . Calnexin served as loading control ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody . While GnT - V Nicastrin maturation were similarly affected by Syntaxin 5 knock down SPPL2c overexpression , SPPL2a maturation was hardly impaired in Syntaxin 5 knock down .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O

C) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT-V, as well maturation of the endogenous glycoproteins Nicastrin (NCT) and SPPL2a was analyzed in Western Blot. Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody. While GnT-V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression, SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells.


D ) Syntaxin 5 localization is impaired by overexpression of SPPL2c . Endogenous Syntaxin 5 was visualized in control without SPPL2c expression ( Ctr ) in overexpressing SPPL2c wt or SPPL2c D / A using immunofluorescence . BiP was used as a marker of ER TGN46 as Golgi marker . arrow indicating with disturbed Golgi Syntaxin 5 . Scale bar 5 µm .

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D) Syntaxin 5 localization is impaired by overexpression of SPPL2c. Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression (Ctr) and in cells overexpressing SPPL2c wt or SPPL2c D/A using immunofluorescence. BiP was used as a marker protein of the ER and TGN46 as Golgi marker. White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining. Scale bar 5 µm.


E ) Syntaxin 5 knock down induces mislocalization of GnT - V similar to SPPL2c overexpression ectopically expressing GnT - V were treated either with non - specific siRNA ( siCtr ) or with siRNA targeting Syntaxin 5 ( siStx5 ) . GnT - V was visualized in immunofluorescence using anti - V5 antibody targeting its C - terminal tag either BIP was co - stained as ER marker or TGN46 as Golgi marker . Scale bar 5 µm .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O

E) Syntaxin 5 knock down induces mislocalization of GnT-V similar to SPPL2c overexpression Cells ectopically expressing GnT-V were treated either with non-specific siRNA (siCtr) or with siRNA targeting Syntaxin 5 (siStx5). GnT-V was visualized in immunofluorescence using anti-V5 antibody targeting its C-terminal tag and either BIP was co-stained as ER marker protein or TGN46 as Golgi marker protein. Scale bar 5 µm.


After seeding were cultured for 72h expression of catalytically active ( wt ) or non - active ( D / A ) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated . Non - transfected ( Ctr ) or non - induced SPPL2c ( n / i ) served as controls . ER was stained with anti - BiP antibody , cis / medial - Golgi with anti - Giantin , trans - Golgi Network with anti - TGN46 antibody Golgi subcompartments with CAB45 - specific antibody in immunofluorescence . Note that only upon expression of catalytically active SPPL2c morphologies of ER cis - Golgi change compared to controls . Scale bar 5 µm .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.


A ) Western Blot of SPPL2c in preparations from HEK293 inducibly overexpressing SPPL2c , human testis mouse testis tissue . For human mouse tissue , 5 μg of each total testis preparation is loaded . fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 is diluted 1 : 50 ( 0 . 1 μg total ) .

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A) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c, human testis and mouse testis tissue. For human and mouse tissue, 5 μg protein of each total testis preparation is loaded. The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1:50 (0.1 μg total protein).


B ) Western Blot of selected glycoproteins in mouse tissues from two wildtype C57BL / 6J ( Black6 ) mice . Endogenous Nicastrin ( NCT ) SPPL2a were analysed in preparations from testis , kidney , brain , spleen liver in littermate mice . Note that immature ( im ) accumulate in testis compared to other tissues . SPPL2c is only detected in testis tissue .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O 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-MEASURED_VAR O O O O O O O

B) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL/6J (Black6) mice. Endogenous Nicastrin (NCT) and SPPL2a were analysed in membrane preparations from testis, kidney, brain, spleen and liver in littermate male mice. Note that immature proteins (im) accumulate in testis compared to other tissues. SPPL2c protein is only detected in testis tissue.


C ) of Syntaxin 8 ( Stx8 ) in SPPL2c - / - mice compared to wildtype ( wt ) . isolates from whole testis were analyzed for Syntaxin 8 ( Stx8 ) SPPL2c expression . TMEM192 serves as loading control .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O

C) Analysis of Syntaxin 8 (Stx8) in SPPL2c-/- mice compared to wildtype (wt) littermates. Membrane isolates from whole testis were analyzed for Syntaxin 8 (Stx8) and SPPL2c expression. TMEM192 serves as loading control.


D ) Syntaxin 8 were quantified from respective Western blots by densitometric normalized to those of TMEM192 . Bars represent mean values of n = 3 ± SD normalized to mean Syntaxin 8 of wildtype mice . Statistical significance was calculated applying an unpaired , two - sided Student ' s t - . * p ≤ 0 . 05 . Note that Syntaxin 8 is accumulating in SPPL2c - / - mice .

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D) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192. Bars represent mean values of n=3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice. Statistical significance was calculated applying an unpaired, two-sided Student's t-test. * p≤0.05. Note that Syntaxin 8 is accumulating in SPPL2c-/- mice.


E ) Real - time qPCR of Syntaxin 8 ( Stx8 ) mRNA in testis of SPPL2c - / - wildtype mice normalized to that of Tuba1a . diagram depicts mean values of Stx8 mRNA normalized to that of wildtype of n = 6 individuals from two independent experiments ± SD . For statistical an unpaired , two - sided Student ' s t - was performed . ns = not significant .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Real-time qPCR analysis of Syntaxin 8 (Stx8) mRNA levels in testis of SPPL2c-/- and the wildtype mice normalized to that of Tuba1a. The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n=6 individuals from two independent experiments ± SD. For statistical analysis an unpaired, two-sided Student's t-test was performed. ns = not significant.


F ) Immunohistochemical stainings of cross - sectioned seminiferous tubules of SPPL2c + / + SPPL2c - / - mice using anti - Cab45 antibody . Stages VII / VIII are depicted . Higher magnifications are indicated with dashed lines black arrows indicate pre - acrosomal structures . Scale bar 50 µm .

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F) Immunohistochemical stainings of cross-sectioned seminiferous tubules of SPPL2c+/+ and SPPL2c-/- mice using anti-Cab45 antibody. Stages VII/VIII are depicted. Higher magnifications are indicated with dashed lines and black arrows indicate pre-acrosomal structures. Scale bar 50 µm.


G ) Volcano plot depicting binding affinities of lectins on sperm lysates from SPPL2c - / - mice compared to SPPL2c + / + ( wt ) . Each circle represents one lectin . lectins above dashed ( blue ) have a significant p - value < 0 . 05 . Lectins on left side of blot demonstrate reduced binding while UEA - I on right side has increased binding . n = 4 per genotype , two - sided , unpaired Student ' s t - .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

G) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c-/- mice compared to SPPL2c+/+ (wt). Each circle represents one lectin. The lectins above the dashed line (light blue) have a significant p-value<0.05. Lectins on the left side of the blot demonstrate reduced binding while UEA-I on the right side has increased binding. n=4 animals per genotype, two-sided, unpaired Student's t-Test.


( A ) GFP - LC3 CHO were preincubated with 1 mM dybutiryl cAMP ( + dbcAMP , panels d , e , f ) for 30 min then were treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) in continuous presence of dbcAMP . without any treatment were used as control ( − dbcAMP , panels a , b , c ) . were immediately analyzed by confocal microscopy . A of of presenting LC3 ( i . e . , stimulated ) upon incubation with different conditions is shown in right panel ( n = 100 / condition , * p < 0 . 05 paired Student ' s t - ) . These data are representative of three independent experiments .

O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP, panels d, e, and f) for 30 min and then were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) in the continuous presence of dbcAMP. Cells without any treatment were used as control (−dbcAMP, panels a, b, and c). Cells were immediately analyzed by confocal microscopy. A quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown in the right panel (n = 100 cells/condition, * p<0.05 paired Student's t-test). These data are representative of three independent experiments.


( B ) GFP - LC3 CHO were incubated with complete medium in absence ( − dbcAMP ) or presence of dbcAMP ( + dbcAMP ) treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) with ( lower panel ) or without ( upper panel ) bafilomycin A1 to block lysosomal degradation . Afterwards , were lysed with sample buffer samples were subjected to Western blot using a rabbit anti - LC3 corresponding HRP - labeled secondary antibody , subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of three independent experiments . ( C ) band intensities of two independent experiments were quantificated with Adobe Photoshop program , normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-LC3 CHO cells were incubated with complete medium in the absence (−dbcAMP) or presence of dbcAMP (+dbcAMP) and treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) with (lower panel) or without (upper panel) bafilomycin A1 to block lysosomal degradation. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of three independent experiments. (C) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


( A ) GFP - LC3 CHO were preincubated for 30 min with 10 µM H89 , a PKA inhibitor , in presence ( panels a , b , c ) or absence ( panels d , e , f ) of 1 mM dbcAMP . Subsequently , they were incubated for 2 h in starvation medium ( Stv , panels b e ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla , panels c f ) in full nutrient media . without any treatment were used as control ( Ctr , panels a d ) . were finally analyzed by confocal microscopy . are representative of three independent experiments .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM H89, a PKA inhibitor, in the presence (panels a, b, and c) or absence (panels d, e, and f) of 1 mM dbcAMP. Subsequently, they were incubated for 2 h in starvation medium (Stv, panels b and e) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels c and f) in full nutrient media. Cells without any treatment were used as control (Ctr, panels a and d). Cells were finally analyzed by confocal microscopy. Images are representative of three independent experiments.


( B ) of of presenting LC3 ( i . e . , stimulated ) upon incubation with different conditions . * * p < 0 . 01 ( paired Student ' s t - , n = 100 / condition ) . These data are representative of two independent experiments .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. ** p<0.01 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.


( C ) GFP - LC3 CHO were preincubated for 30 min in presence or absence of 10 µM H89 , then they were incubated for 2 h with 1 mM dbcAMP to verify activity of H89 . Afterwards , were lysed with sample buffer samples were subjected to Western blot using a rabbit anti - phosphoCREB corresponding HRP - labeled secondary antibody , subsequently developed with an enhanced chemiluminescence detection kit . band intensities were quantified with Adobe Photoshop program ( lower panel ) . These data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) GFP-LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89, and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-phosphoCREB and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantified with the Adobe Photoshop program (lower panel). These data are representative of two independent experiments.


( A ) GFP - LC3 CHO were preincubated with 10 µM 8 - pCPT - 2 ′ - O - Me - cAMP ( 8 - pCPT ) for 30 min then they were treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) . without any treatment were used as control . were immediately analyzed by confocal microscopy . of of presenting LC3 upon incubation under different conditions is shown in right panel ( n = 50 / condition ) . These data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GFP-LC3 CHO cells were preincubated with 10 µM 8-pCPT-2′-O-Me-cAMP (8-pCPT) for 30 min and then they were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv). Cells without any treatment were used as control. Cells were immediately analyzed by confocal microscopy. Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel (n = 50 cells/condition). These data are representative of two independent experiments.


( B ) CHO were cotransfected with GFP - LC3 myc - EPAC wt ( left panel ) or myc - ΔEPAC ( right panel ) . Twenty - four hours after transfection they were incubated for 2 h in starvation medium ( Stv ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . without any treatment were used as control ( Ctr ) . were analyzed by confocal microscopy . are representative of two independent experiments . ( C ) of of presenting LC3 upon incubation under different conditions ( n = 20 / condition ) . These data are representative of two independent

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(B) CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (left panel) or myc-ΔEPAC (right panel). Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (C) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions (n = 20 cells/condition). These data are representative of two independent experiments


( A ) CHO were cotransfected with RFP - LC3 GFP - Rap2b wt ( left panel ) or GFP - Rap2b ΔAAX ( right panel ) . Twenty - four hours after transfection , they were incubated for 2 h in starvation medium ( Stv ) , with 50 ng / µl of rapamycin ( Rapa ) in full nutrient media , or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . without any treatment were used as control ( Ctr ) . were analyzed by confocal microscopy . are representative of three independent experiments .

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(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (left panel) or GFP-Rap2b ΔAAX (right panel). Twenty-four hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) in full nutrient media, or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of three independent experiments.


( B ) of of presenting LC3 ( i . e . , stimulated ) upon incubation with different conditions is shown . * p < 0 . 05 , * * p < 0 . 01 ( paired Student ' s t - , n = 50 / condition ) . These data are representative of three independent experiments .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.


( C ) CHO were transfected with GFP - Rap2b wt or GFP - Rap2b ΔAAX incubated with complete medium without ( Ctr ) or with 10 µg / ml of α - hemolysin ( Hla ) for 4 h , with 50 ng / µl of rapamycin ( Rapa ) or subjected to starvation conditions ( Stv ) for 2 h in absence or presence of bafilomycin A1 . Afterwards , were lysed with sample buffer samples were subjected to Western blot using a rabbit anti - LC3 corresponding HRP - labeled secondary antibody , subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of two independent experiments .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(C) CHO cells were transfected with GFP-Rap2b wt or GFP-Rap2b ΔAAX and incubated with complete medium without (Ctr) or with 10 µg/ml of α-hemolysin (Hla) for 4 h, with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h in the absence or presence of bafilomycin A1. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.


( D ) of Western blot bands intensities of two independent experiments with Adobe Photoshop program .

(D) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program.


( A ) HeLa were cotransfected with Rap2b siRNA GFP - LC3 ( right panel ) or irrelevant siRNA GFP - LC3 ( left panel ) . Forty - eight hours after transfection , they were incubated for 2 h in starvation medium ( Stv ) , with 50 ng / µl of rapamycin ( Rapa ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . without any treatment were used as control ( Ctr ) . were analyzed by confocal microscopy . are representative of two independent experiments .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells were cotransfected with Rap2b siRNA and GFP-LC3 (right panel) or irrelevant siRNA and GFP-LC3 (left panel). Forty-eight hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments.


( B ) of of presenting LC3 upon incubation under different conditions . Data correspond to two independent experiments ( n = 50 / condition ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions. Data correspond to two independent experiments (n = 50 cells/condition).


( C ) Upper panel : knockdown of Rap2b was determined by Western blot as indicated in Materials Methods . Lower panel : band intensities of two independent experiments were quantified with Adobe Photoshop program , normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Upper panel: The knockdown of Rap2b was determined by Western blot as indicated in Materials and Methods. Lower panel: The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


( D ) HeLa were cotransfected with GFP - LC3 Rap2b siRNA or an irrelevant siRNA incubated for 4 h with complete medium in absence ( Ctr ) or presence of 10 µg / ml of α - hemolysin ( Hla ) , with 50 ng / µl of rapamycin ( Rapa ) or subjected to starvation conditions ( Stv ) for 2 h . Afterwards , were lysed with sample buffer samples were subjected to Western blot using a rabbit anti - LC3 corresponding HRP - labeled secondary antibody , subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of two independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(D) HeLa cells were cotransfected with GFP-LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence (Ctr) or presence of 10 µg/ml of α-hemolysin (Hla), with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.


( E ) band intensities of two independent experiments were quantified with Adobe Photoshop program , normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - ) .

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

(E) The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


cAMP cannot inhibit autophagy induced by toxin in overexpressing Rap2b negative mutant . ( A ) CHO were cotransfected with RFP - LC3 GFP - Rap2b ΔAAX . Twenty - four hours after transfection they were incubated for 2 h in starvation medium ( Stv ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) in presence ( right panels ) or absence of 1 mM dbcAMP ( left panels ) . without any treatment were used as control ( Ctr ) . were analyzed by confocal microscopy . are representative of two independent experiments . ( B ) of of presenting LC3 ( i . e . , stimulated ) upon incubation with different conditions . These data are representative of two independent experiments .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.


( A ) GFP - LC3 CHO were preincubated for 30 min with 10 µM of calpeptin ( a calpain inhibitor , panels b , f , j ) , with 1 mM dbcAMP ( panels d , h l ) , or with Calpeptin + dbcAMP ( panels c , g , k ) . Afterwards , they were incubated for 2 h in starvation medium ( Stv , panels e , f , g , h ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla , panels i , j , k , l ) . without any treatment were used as control ( Ctr , panels a , b , c d ) . were analyzed by confocal microscopy . are representative of two independent experiments . ( B ) of of presenting LC3 ( i . e . , stimulated ) upon incubation with different conditions . * p < 0 . 05 ( paired Student ' s t - , n = 100 / condition ) . These data are representative of two independent experiments .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin (a calpain inhibitor, panels b, f, and j), with 1 mM dbcAMP (panels d, h and l), or with Calpeptin+dbcAMP (panels c, g, and k). Afterwards, they were incubated for 2 h in starvation medium (Stv, panels e, f, g, and h) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels i, j, k, and l). Cells without any treatment were used as control (Ctr, panels a, b, c and d). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. * p<0.05 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.


( C ) Upper panel : CHO were preincubated for 30 min with 10 µM of calpeptin , with 1 mM dbcAMP , or with calpeptin + dbcAMP , then they were incubated for 4 h in complete medium in presence ( Hla ) or absence ( Ctr ) of α - hemolysin . Afterwards , were lysed with sample buffer samples were subjected to Western blot using a rabbit anti - LC3 corresponding HRP - labeled secondary antibody . bands were subsequently developed with an enhanced chemiluminescence detection kit . Lower panel : of band intensities with Adobe Photoshop program . * p < 0 . 05 ( paired Student ' s t - ) . These data are representative of three independent experiments .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Upper panel: CHO cells were preincubated for 30 min with 10 µM of calpeptin, with 1 mM dbcAMP, or with calpeptin+dbcAMP, and then they were incubated for 4 h in complete medium in the presence (Hla) or absence (Ctr) of α-hemolysin. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. Lower panel: Quantification of the band intensities with the Adobe Photoshop program. * p<0.05 (paired Student's t-test). These data are representative of three independent experiments.


( A ) GFP - LC3 CHO were preincubated with 1 mM dybutiryl cAMP ( + dbcAMP ) for 30 min then were infected for 4 h in presence of db cAMP with wt of S . aureus ( wt ) , mutant deficient for α - hemolysin ( Hla − ) , or Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . without any treatment were used as control ( − dbcAMP ) . nucleus were labeled with TOPRO as indicated in Materials Methods , immediately visualized by confocal microscopy . are representative of two independent experiments .

O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−) +pHla). Cells without any treatment were used as control (−dbcAMP). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods, and immediately visualized by confocal microscopy. Images are representative of two independent experiments.


. ( B ) of of presenting LC3 ( i . e . , stimulated ) upon incubation in different conditions . * p < 0 . 05 , * * p < 0 . 01 ( paired Student ' s t - , n = 50 / condition ) . These data are representative of two independent experiments .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation in the different conditions. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of two independent experiments.


( C ) of number of CFU / ml of CHO infected for 3 h with wt of S . aureus , mutant deficient for α - hemolysin ( Hla − ) , or Hla ( − ) mutant expressing an α - hemolysin plasmid in absence or presence of cAMP . * p < 0 . 05 ( paired Student ' s t - ) . These data are representative of two independent experiments .

O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the number of CFU/ml of CHO cells infected for 3 h with the wt strain of S. aureus, the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid in the absence or presence of cAMP. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.


CHO were cotransfected with GFP - LC3 myc - EPAC wt ( upper panel ) or myc - ΔEPAC ( lower panel ) . Twenty - four hours after transfection , they were infected for 4 h with wt of S . aureus ( wt ) , mutant deficient for α - hemolysin ( Hla − ) , or Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . nucleus were labeled with TOPRO as indicated in Materials Methods immediately visualized by confocal microscopy . are representative of three independent experiments .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (upper panel) or myc-ΔEPAC (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. Images are representative of three independent experiments.


B ) of of presenting LC3 upon infection . These data are representative of three independent experiments .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O

B) Quantification of the percentage of cells presenting LC3 puncta upon the infection. These data are representative of three independent experiments.


( C ) of of decorated with EPAC in infected with S . aureus . * * p < 0 . 01 ( paired Student ' s t - , n = 20 / condition ) . These data are representative of three independent experiments .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the percentage of bacteria decorated with EPAC in cells infected with S. aureus. ** p<0.01 (paired Student's t-test, n = 20 cells/condition). These data are representative of three independent experiments.


( A ) CHO were cotransfected with RFP - LC3 GFP - Rap2b wt ( upper panel ) or GFP - Rap2b ΔAAX ( lower panel ) . Twenty - four hours after transfection , they were infected for 4 h with wt of S . aureus ( wt ) , mutant deficient for α - hemolysin ( Hla − ) , or Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . nucleus were labeled with TOPRO as indicated in Materials Methods immediately visualized by confocal microscopy . These data are representative of three independent experiments .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (upper panel) or GFP-Rap2b ΔAAX (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. These data are representative of three independent experiments.


( B ) of of infected presenting LC3 upon infection . * p < 0 . 05 , * * * p < 0 . 001 ( paired Student ' s t - , n = 50 / condition ) . These data are representative of three independent experiments .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection. * p<0.05, *** p<0.001 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.


( C ) of of decorated with Rap2b wt upon infection . * * p < 0 . 01 ( paired Student ' s t - , n = 30 / condition ) . These data are representative of three independent

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection. ** p<0.01 (paired Student's t-test, n = 30 cells/condition). These data are representative of three independent experim


( A ) HeLa were incubated for 4 h with complete medium in presence ( Hla ) or absence ( Ctr ) of 10 µg / ml α - hemolysin or they were infected for 4 h with wt of S . aureus ( wt ) or α - hemolysin deficient mutant ( Hla − ) . Afterwards , were lysed with sample buffer samples were subjected to Western blot using a rabbit anti - Rap2b corresponding HRP - labeled secondary antibody . bands were subsequently developed with an enhanced chemiluminescence detection kit . A of bands intensities with Adobe Photoshop program is shown in lower panel . These data are representative of two independent experiments .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel. These data are representative of two independent experiments.