( B ) Replication of GFP - expressing T . gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging . Since vacuoles containing more than eight tachyzoites are not always easy to score , we tallied vacuoles above eight as being 8 + in size . At 36 hours , both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites ( three or more doublings ) . Vacuoles in parabulin - treated cultures contain significantly fewer parasites , indicating that replication is slowed . The graph shows three biological replicates with values normalized to 100 % for each condition and each replicate , error bars represent standard error of the mean between experiments .

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(B) Replication of GFP-expressing T. gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging. Since vacuoles containing more than eight tachyzoites are not always easy to score, we tallied vacuoles above eight as being 8+ in size. At 36 hours, both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites (three or more doublings). Vacuoles in parabulin-treated cultures contain significantly fewer parasites, indicating that replication is slowed. The graph shows three biological replicates with values normalized to 100% for each condition and each replicate, error bars represent standard error of the mean between experiments.


( C ) The parabulin EC50 value for T . gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions . Error bars represent standard error of the mean . The results represent the average of 18 readings ( 6 biological replicates with 3 technical replicates of each treatment ) ± standard error of the mean .

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(C) The parabulin EC50 value for T. gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions. Error bars represent standard error of the mean. The results represent the average of 18 readings (6 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.


( D ) Phase contrast of extracellular parasite cultures . Serial passage of T . gondii in 40 - 80 μM parabulin induces accumulation of aberrant tachyzoite forms . These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding .

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(D) Phase contrast of extracellular parasite cultures. Serial passage of T. gondii in 40-80 μM parabulin induces accumulation of aberrant tachyzoite forms. These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding.


A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP - positive cells was determined 16 h later by flow cytometry .

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A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP-positive cells was determined 16 h later by flow cytometry.


B LDH release from HEK293T cells transfected with the pRetroX TETOne3G - eGFP plasmid only ( vector ) or pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD . At 24 h post transfection , cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined . Graphs show mean and s . d . of quadruplicate wells .

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B LDH release from HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. At 24 h post transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells.


C - D . PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G - eGFP harbouring the N - terminal fragment of GSDMD in the presence of osmoprotectants . At 24 h post transfection , PEGs of the indicated molecular weights were added to a final concentration of 30 mM , cells were treated with 250 ng / ml doxycycline for 8 h and the level of PI staining ( C ) or LDH release ( D ) was determined .

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C-D. PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng/ml doxycycline for 8 h and the level of PI staining (C) or LDH release (D) was determined.


E - F . PI staining of and LDH release from LPS - primed primary BMDMs infected with log - phase S . typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight ( numbers on the X - axis , 30 mM final concentration ) . * p < 0 . 05 as determined by student ' s t test . Graphs show mean and s . d . of quadruplicate wells . Data are representative of at least three independent expeirments .

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E-F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight (numbers on the X-axis, 30 mM final concentration).* p<0.05 as determined by student's t test. Graphs show mean and s.d. of quadruplicate wells. Data are representative of at least three independent expeirments.


A . Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD , cleaved GSDMD and α - tubulin in the cell lysates of immortalized LPS - primed WT macrophages left uninfected ( NS ) or infected for 10 - 40 minutes with log phase S . typhimurium ( MOI = 50 ) .

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A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10-40 minutes with log phase S. typhimurium (MOI=50).


C . Fractionation and immunoblot analysis for GSDMD , Na + K + ATPase , VDAC ( Voltage - dependent anion channel ) , HDAC1 ( Histone deacetylase 1 ) and GAPDH ( Glycerinaldehyd - 3 - phosphat - Dehydrogenase ) of WT macrophages infected for 10 and 20 minutes with log phase S . typhimurium ( MOI = 50 ) . Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane .

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C. Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (Voltage-dependent anion channel), HDAC1 (Histone deacetylase 1) and GAPDH (Glycerinaldehyd-3-phosphat-Dehydrogenase) of WT macrophages infected for 10 and 20 minutes with log phase S. typhimurium (MOI=50). Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane.


D . Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S . typhimurium ( MOI = 50 ) . Extraction was carried in variable conditions as described in the Material and Methods section .

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D. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S. typhimurium (MOI=50). Extraction was carried in variable conditions as described in the Material and Methods section.


A . Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . The protein was cleaved in a time - dependent manner into two bands of 31 kDa ( GSDMDNterm ) and 22 kDa ( GSDMDCterm ) .

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A. Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. The protein was cleaved in a time-dependent manner into two bands of 31 kDa (GSDMDNterm) and 22 kDa (GSDMDCterm).


B . Cross - linking experiment of full - length and cleaved GSDMD . GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase - 1 . After enzymatic cleavage , GSDMDNterm is highly cross - linked by DSS , resulting in the gel - impenetrating species highlighted by the arrow . GSDMDCterm is not cross - linked .

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B. Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked.


C . GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase - 1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E . coli . After 2 h , the lipid fraction ( L ) was separated from the supernatant ( S ) by ultracentrifugation at 4C for 1 h at 120 , 000g .

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C. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4C for 1 h at 120,000g.


A - I . Dye release time courses from liposomes as a percentage of maximal release . A . Five different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli polar lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 130 , 100 , 65 ( colored dark to light orange ) . The time point of 20 min is highlighted by a vertical dashed line .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. A. Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli polar lipid extract were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . B . Five different reactions , where 130 nM of GSDMD and 400μM 6 - carboxifluorescein - loaded liposomes prepared with E . Coli extract polar lipid were incubated with caspase - 1 concentration of ( nM ) : 15 , 8 , 5 , 2 . 5 , 1 . 2 ( colored dark to light blue ) . The time point of 20 min is highlighted by a vertical dashed line .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. B. Five different reactions, where 130 nM of GSDMD and 400μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli extract polar lipid were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . C . Three different reactions , where 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of ( nM ) : 520 , 260 , 100 ( colored dark to light green ) .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. C. Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of (nM): 520, 260,100 (colored dark to light green).


A - I . Dye release time courses from liposomes as a percentage of maximal release . D . Dye release at 20 minutes reaction as a function of GSDMD ( dark orange ) and caspase - 1 ( dark blue ) concentrations . Errors bars for three independent experiments are shown .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. D. Dye release at 20 minutes reaction as a function of GSDMD (dark orange) and caspase-1 (dark blue) concentrations. Errors bars for three independent experiments are shown.


A - I . Dye release time courses from liposomes as a percentage of maximal release . E . Two different sets of reactions , where wild type GSDMD ( dark to light orange ) and the mutant GSDMDI104N ( dark to light blue ) were independently incubated at the concentrations of 260 , 130 and 65 nM with 5 nM caspase - 1 and 400 μM 6 - carboxifluorescein - loaded liposomes . The time point of 60 minutes is highlighted by a vertical dashed line .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. E. Two different sets of reactions, where wild type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130 and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes. The time point of 60 minutes is highlighted by a vertical dashed line.


A - I . Dye release time courses from liposomes as a percentage of maximal release . F . Dye release at 60 minutes of reaction as a function of GSDMD wild type ( dark orange ) and GSDMDI104N ( dark blue ) concentration . Error bars for three independent experiments are shown .

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A-I. Dye release time courses from liposomes as a percentage of maximal release. F. Dye release at 60 minutes of reaction as a function of GSDMD wild type (dark orange) and GSDMDI104N (dark blue) concentration. Error bars for three independent experiments are shown.


A - I . Dye release time courses from liposomes as a percentage of maximal release . G - I . Dye release from 400 μM liposomes loaded with the 6 - carboxifluorescein - derivates FD - 20 , FD - 40 and FD - 150 , with variable Stokes diameters , as indicated . 130 nM of GSDMD and 5 nM caspase - 1 were incubated with the liposomes .

A-I. Dye release time courses from liposomes as a percentage of maximal release. G-I. Dye release from 400 μM liposomes loaded with the 6-carboxifluorescein-derivates FD-20, FD-40 and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes.


A - C . Cryo - EM micrographs of GSDMDNterm pores in E . coli extract polar lipids liposomes . The micrographs were acquired at protein / lipid molar ratios of 1 / 1000 , 1 / 500 and 1 / 100 , respectively . Black arrows indicate ring - shaped structures corresponding to oligomeric GSDMDNterm pore forms . Scale bars = 80 nm .

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A-C. Cryo-EM micrographs of GSDMDNterm pores in E. coli extract polar lipids liposomes. The micrographs were acquired at protein/lipid molar ratios of 1/1000, 1/500 and 1/100, respectively. Black arrows indicate ring-shaped structures corresponding to oligomeric GSDMDNterm pore forms. Scale bars = 80 nm.


D . Proteoliposome with protein / lipid molar ratio of 1 / 100 at higher magnification . Scale bar = 80 nm .

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D. Proteoliposome with protein/lipid molar ratio of 1/100 at higher magnification. Scale bar = 80 nm.


A . AFM topograph of GSDMDNterm bound to lipid membranes of E . coli extract polar lipids . Overview topograph showing arc - , slit - and ring - like GSDMD oligomers .

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A. AFM topograph of GSDMDNterm bound to lipid membranes of E. coli extract polar lipids. Overview topograph showing arc-, slit- and ring-like GSDMD oligomers.


B - E . High - resolution AFM topographs and height profiles of GSDMDNterm oligomers ( red curves ) . Height profiles were measured along the red lines in the topographs . Dashed zero lines ( 0 nm ) indicate the surface of the lipid membrane ; heights of < − 4 nm indicate that the oligomers formed transmembrane pores . Topographs were taken in buffer solution ( 150 NaCl , 20 mM Hepes , pH 7 . 8 ) at room temperature . The experiment was reproduced at least three times using independent liposome and GSDMD preparations .

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B-E. High-resolution AFM topographs and height profiles of GSDMDNterm oligomers (red curves). Height profiles were measured along the red lines in the topographs. Dashed zero lines (0 nm) indicate the surface of the lipid membrane; heights of <−4 nm indicate that the oligomers formed transmembrane pores. Topographs were taken in buffer solution (150 NaCl, 20 mM Hepes, pH 7.8) at room temperature. The experiment was reproduced at least three times using independent liposome and GSDMD preparations.


F - H . Analysis of GSDMDNterm oligomers bound to lipid membranes of E . coli extract polar lipids . F . GSDMDNterm oligomers assembled into arcs , slits and rings leading to transmembrane ( TM ) pores or not transmembrane aggregate ( n = 218 ) . G . Height of GSDMDNterm oligomers protruding from the lipid membrane . Bars represent mean and error bars represent s . e . m . ( n = 218 ) . H . Distribution of the diameters of rings formed by GSDMDNterm oligomers . The average distribution was 21 . 2 ± 5 . 6 nm ( n = 164 ; average ± s . d . ) and the bin size was 3 nm .

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F-H. Analysis of GSDMDNterm oligomers bound to lipid membranes of E. coli extract polar lipids. F. GSDMDNterm oligomers assembled into arcs, slits and rings leading to transmembrane (TM) pores or not transmembrane aggregate (n = 218). G. Height of GSDMDNterm oligomers protruding from the lipid membrane. Bars represent mean and error bars represent s.e.m. (n = 218). H. Distribution of the diameters of rings formed by GSDMDNterm oligomers. The average distribution was 21.2 ± 5.6 nm (n = 164; average ± s.d.) and the bin size was 3 nm.


Co - immunoprecipitation assays in HEK293T cells after co - transfection with Flag - TRIM27 and the indicated combinations of empty HA - vector , HA - tagged FIP200 , ULK1 , or ATG13 before immunoprecipitation with anti - HA antibodies . Immunoprecipitates ( top panel ) and input samples ( bottom panel ) were then subjected to Western blotting against anti - HA or Flag . Data information : the experiments were performed at least three times independently with similar results .

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Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and the indicated combinations of empty HA-vector, HA-tagged FIP200, ULK1, or ATG13 before immunoprecipitation with anti-HA antibodies. Immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to Western blotting against anti-HA or Flag. Data information: the experiments were performed at least three times independently with similar results.


C - F . WT ( C ) , FIP200 knockout ( KO ) ( D ) , ULK1 / 2 knockout ( E ) MEF cells or ATG13 knockout 4T1 cells ( F ) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G ( IgG ) or anti - TRIM27 before blotting against the indicated endogenous forms of TRIM27 , FIP200 , ULK1 , ATG13 , and USP7 . Data information : the experiments were performed at least three times independently with similar results .

C-F. WT (C), FIP200 knockout (KO) (D), ULK1/2 knockout (E) MEF cells or ATG13 knockout 4T1 cells (F) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G (IgG) or anti-TRIM27 before blotting against the indicated endogenous forms of TRIM27, FIP200, ULK1, ATG13, and USP7. Data information: the experiments were performed at least three times independently with similar results.


G . Co - immunoprecipitation assays in HEK293T cells after co - transfection with Flag - TRIM27 and either full length ULK ( HA - ULK1 1 - 1051 ) or the indicated various truncated forms of ULK1 designated HA - ULK1 1 - 828 , HA - ULK1 1 - 600 , and HA - ULK1 1 - 500 ; AA , amino acids . Data information : , the experiments were performed at least three times independently with similar results .

G. Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and either full length ULK (HA-ULK1 1-1051) or the indicated various truncated forms of ULK1 designated HA-ULK1 1-828, HA-ULK1 1-600, and HA-ULK1 1-500; AA, amino acids. Data information: , the experiments were performed at least three times independently with similar results.


H . Co - immunoprecipitation assays in HeLa cells stably expressing HA - ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated . Data information : the experiments were performed at least three times independently with similar results .

H. Co-immunoprecipitation assays in HeLa cells stably expressing HA-ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated. Data information: the experiments were performed at least three times independently with similar results.


HeLa cells stably expressing Flag - TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium . Anti - Flag immunoprecipitates ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against Flag and ULK1 . Data information : the experiments were performed at least three times independently with similar results .

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HeLa cells stably expressing Flag-TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium. Anti-Flag immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to blotting against Flag and ULK1. Data information: the experiments were performed at least three times independently with similar results.


Purified recombinant GST or GST - TRIM27 was incubated with recombinant HA - ULK1 and bound ULK1 analyzed by Western blotting . Data information : the experiments were performed at least three times independently with similar results .

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Purified recombinant GST or GST-TRIM27 was incubated with recombinant HA-ULK1 and bound ULK1 analyzed by Western blotting. Data information: the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T cells after co - transfection with HA - ULK1 and His - Ub along with Flag - TRIM27 ( A ) Samples recovered with Ni - NTA ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against anti - HA , Flag or His as indicated . Data information : , the experiments were performed at least three times independently with similar results .

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Ubiquitination assays in HEK293T cells after co-transfection with HA-ULK1 and His-Ub along with Flag-TRIM27 (A) Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA, Flag or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs ( shTRIM27 - 1 and - 2 ) ( B ) . Samples recovered with Ni - NTA ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against anti - HA or His as indicated . Data information : , the experiments were performed at least three times independently with similar results .

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Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs (shTRIM27-1 and -2) (B). Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


C . In vitro ubiquitination assays performed with the indicated combinations of recombinant GST , GST - TRIM27 , affinity - purified HA - ULK1 and His - Ub in the presence or absence of E1 , E2 , and ATP . The reactions were analyzed with anti - Ub , anti - HA , or anti - GST . Data information : the experiments were performed at least three times independently with similar results .

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C. In vitro ubiquitination assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1 and His-Ub in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST. Data information: the experiments were performed at least three times independently with similar results.


Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs ( D ) Data information : , the experiments were performed at least three times independently with similar results .

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Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs (D) Data information: , the experiments were performed at least three times independently with similar results.


Western blot analysis of HEK293T cells transfected with empty Flag vector ( - ) or Flag - TRIM27 after treatment with or without 10 μM MG132 for 4 hours ( E ) . Data information : the experiments were performed at least three times independently with similar results .

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Western blot analysis of HEK293T cells transfected with empty Flag vector (-) or Flag-TRIM27 after treatment with or without 10 μM MG132 for 4 hours (E). Data information: the experiments were performed at least three times independently with similar results.


F . Fluorescence microscopy image of MEFs with stable expression of mCherry - ULK1 and GFP - TRIM27 after treating cells with DMSO ( MOCK ) or 10 μM MG132 for 4 hours . Scale bar , 10 μm . Data information : the experiments were performed at least three times independently with similar results .

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F. Fluorescence microscopy image of MEFs with stable expression of mCherry-ULK1 and GFP-TRIM27 after treating cells with DMSO (MOCK) or 10 μM MG132 for 4 hours. Scale bar, 10 μm. Data information: the experiments were performed at least three times independently with similar results.


HA - ULK1 levels in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T cells with or without co - expression of Flag - TRIM27 ( G , H ) Western blots against HA - ULK1 and actin control ( G , were subjected to densitometric quantitation ( H , Data information : the experiments were performed at least three times independently with similar results . All the statistical data are presented as mean ± s . d . of three independent experiments . Statistical analysis was performed using two - tailed unpaired Student ' s t - tests ( H , * * P < 0 . 01 .

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HA-ULK1 levels in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells with or without co-expression of Flag-TRIM27 (G, H) Western blots against HA-ULK1 and actin control (G, were subjected to densitometric quantitation (H, Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests (H , **P < 0.01.


endogenous ULK1 levels ( I , J ) in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T cells or without TRIM27 RNAi ( I , J ) . and actin control I ) were subjected to densitometric quantitation J ) . Data information : the experiments were performed at least three times independently with similar results . All the statistical data are presented as mean ± s . d . of three independent experiments . Statistical analysis was performed using two - tailed unpaired Student ' s t - tests J ) , * * P < 0 . 01 .

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endogenous ULK1 levels (I, J) in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells or without TRIM27 RNAi (I, J). and actin control I) were subjected to densitometric quantitation J). Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests J), **P < 0.01.


Ubiquitination assays were performed in HEK293T cells after co - transfection with full length HA - ULK1 or the indicated truncated constructs ( K ) in the presence or absence of Flag - TRIM27 . Data information : , the experiments were performed at least three times independently with similar results .

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Ubiquitination assays were performed in HEK293T cells after co-transfection with full length HA-ULK1 or the indicated truncated constructs (K) in the presence or absence of Flag-TRIM27. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays were performed in HEK293T cells after co - transfection with double ( K568 / K571R ) or single ( K568R , K571R ) lysine substitution mutants ( L ) in the presence or absence of Flag - TRIM27 . Data information : the experiments were performed at least three times independently with similar results .

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Ubiquitination assays were performed in HEK293T cells after co-transfection with double (K568/K571R) or single (K568R, K571R) lysine substitution mutants (L) in the presence or absence of Flag-TRIM27. Data information: the experiments were performed at least three times independently with similar results.


M . Ubiquitination of HA - ULK1 in cells co - expressing Flag - TRIM27 with either His - Ub WT , His - Ub K48 , or His - Ub K63 , respectively . Data information : the experiments were performed at least three times independently with similar results .

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M. Ubiquitination of HA-ULK1 in cells co-expressing Flag-TRIM27 with either His-Ub WT, His-Ub K48, or His-Ub K63, respectively. Data information: the experiments were performed at least three times independently with similar results.


N . In vitro ubiquitination assays against affinity - purified HA - ULK1 WT or the HA - ULK1 K568 / K571R mutant in the presence or absence of GST - TRIM27 . Data information : the experiments were performed at least three times independently with similar results .

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N. In vitro ubiquitination assays against affinity-purified HA-ULK1 WT or the HA-ULK1 K568/K571R mutant in the presence or absence of GST-TRIM27. Data information: the experiments were performed at least three times independently with similar results.


O . ULK1 / 2 knockout MEFs stably expressing the indicated HA - ULK1 constructs were subjected to Western blot analysis against HA or actin before ( 0h ) and after treatment with CHX for 12 hours . Data information : the experiments were performed at least three times independently with similar results .

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O. ULK1/2 knockout MEFs stably expressing the indicated HA-ULK1 constructs were subjected to Western blot analysis against HA or actin before (0h) and after treatment with CHX for 12 hours. Data information: the experiments were performed at least three times independently with similar results.


A . Western blot analysis of autophagy marker proteins ( p62 and Ser318 phosphorylated ATG13 ) in WT HeLa cells and two independent TRIM27 KO clones . ULK1 and TRIM27 levels were measured , along with actin as a loading control .

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A. Western blot analysis of autophagy marker proteins (p62 and Ser318 phosphorylated ATG13) in WT HeLa cells and two independent TRIM27 KO clones. ULK1 and TRIM27 levels were measured, along with actin as a loading control.


B . TRIM27 KO HeLa cells were reconstituted with Flag - TRIM27 and Western blotting was performed as per ( A ) .

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B. TRIM27 KO HeLa cells were reconstituted with Flag-TRIM27 and Western blotting was performed as per (A).


C . LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 ( BAF A1 ) .

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C. LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 (BAF A1).


D . Western blot analysis of indicated proteins in MEFs generated from Trim27 + / + or Trim27 - / - mice .

D. Western blot analysis of indicated proteins in MEFs generated from Trim27 +/+ or Trim27 -/- mice.


E - H . Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27 + / + or Trim27 - / - mice cultured in fed ( E ) or starved conditions ( G ) . Quantification of LC3B puncta Fed conditions ( F ) , starved conditions ( H ) ( n = 50 cells per condition ) . Data are mean ± s . d . in F and H . Scale bar , 10 μm . Data information : ( F , H , Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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E-H. Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27+/+ or Trim27-/- mice cultured in fed (E) or starved conditions (G). Quantification of LC3B puncta Fed conditions (F), starved conditions (H) (n = 50 cells per condition). Data are mean ± s.d. in F and H. Scale bar, 10 μm. Data information: (F, H, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


I - J . Western blot analysis of autophagy markers in the kidneys of three - month - old Trim27 + / + and Trim27 - / - mice . Each lane represents a different mouse ( I ) . Quantification of p62 , LC3 - II / LC3 - I ratio , and LC3 - II / actin ratio in I ( J ) . Data are mean ± s . e . m . for three mice per genotype . Data information : J , Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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I-J. Western blot analysis of autophagy markers in the kidneys of three-month-old Trim27 +/+ and Trim27-/- mice. Each lane represents a different mouse (I). Quantification of p62, LC3-II/LC3-I ratio, and LC3-II/actin ratio in I (J). Data are mean ± s.e.m. for three mice per genotype. Data information: J, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


K . Microscopy analysis of GFP - LC3 puncta in MEFs generated from indicated genotype mice in normal or starvation conditions . Scale bar , 10 μm . L - M . Quantitation of data presented in ( K ) shows mean number of GFP - LC3 puncta per cell ± s . d . ( n = 50 cells per condition ) . Data information : L and M ) Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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K. Microscopy analysis of GFP-LC3 puncta in MEFs generated from indicated genotype mice in normal or starvation conditions. Scale bar, 10 μm. L-M. Quantitation of data presented in (K) shows mean number of GFP-LC3 puncta per cell ± s.d. (n = 50 cells per condition). Data information: L and M) Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


A . Ubiquitination analysis of HA - ULK1 in HeLa derivatives co - expressing His - Ub with or without TRIM27 knockout , the cells were treated with starvation for the indicated time .

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A. Ubiquitination analysis of HA-ULK1 in HeLa derivatives co-expressing His-Ub with or without TRIM27 knockout, the cells were treated with starvation for the indicated time.


B . Western blot analysis of ULK1 levels in HeLa cells with or without TRIM27 knockout . The cells were treated with starvation for the indicated time . The lysates were analyzed using anti - ULK1 , anti - TRIM27 , or anti - Actin .

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B. Western blot analysis of ULK1 levels in HeLa cells with or without TRIM27 knockout. The cells were treated with starvation for the indicated time. The lysates were analyzed using anti-ULK1, anti-TRIM27, or anti-Actin.


C . HA - ULK1 , HA - ULK1 K568 / K571R were stably expressed in ULK1 knockout HeLa cells , the cells were treated with starvation , the lysates were analyzed by western blot using anti - HA , or anti - Actin .

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C. HA-ULK1, HA-ULK1 K568/K571R were stably expressed in ULK1 knockout HeLa cells, the cells were treated with starvation, the lysates were analyzed by western blot using anti-HA, or anti-Actin.


E . Co - immunoprecipitation of Flag - TRIM27 with HA - ULK1 in HEK293T cells with or without STK38L knockdown , the cells were treated with starvation . Flag - TRIM27 was immunoprecipitated using anti - Flag , the bound HA - ULK1 was detected using anti - HA .

E. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 in HEK293T cells with or without STK38L knockdown, the cells were treated with starvation. Flag-TRIM27 was immunoprecipitated using anti-Flag, the bound HA-ULK1 was detected using anti-HA.


F . Co - immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa cells .

F. Co-immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa cells.


G . Co - immunoprecipitation of Flag - STK38L with ULK1 , or TRIM27 in HEK293T cells with or without starvation treatment . Flag - STK38L was immunoprecipitated using anti - Flag , the immunoprecipitates and lysates were analyzed with anti - ULK1 , anti - TRIM27 .

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G. Co-immunoprecipitation of Flag-STK38L with ULK1, or TRIM27 in HEK293T cells with or without starvation treatment. Flag-STK38L was immunoprecipitated using anti-Flag, the immunoprecipitates and lysates were analyzed with anti-ULK1, anti-TRIM27.


H . Co - immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa cells with or without STK38L knockout . The cells were treated with starvation before lysis .

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H. Co-immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa cells with or without STK38L knockout. The cells were treated with starvation before lysis.


I . Western blot analysis of ULK1 levels in starved HeLa cells expressing either Myc - STK38L , or Flag - TRIM27 , or both . The lysates were analyzed using anti - ULK1 , anti - Flag , anti - Myc , or anti - actin .

I. Western blot analysis of ULK1 levels in starved HeLa cells expressing either Myc-STK38L, or Flag-TRIM27, or both. The lysates were analyzed using anti-ULK1, anti-Flag, anti-Myc, or anti-actin.


J . Ubiquitination analysis of HA - ULK1 in HEK293T cells co - expressing Flag - TRIM27 and His - Ub , or together with Myc - STK38L .

J. Ubiquitination analysis of HA-ULK1 in HEK293T cells co-expressing Flag-TRIM27 and His-Ub, or together with Myc-STK38L.


K . Western blot analysis of ULK1 levels in HeLa cells with or without STK38L knockout , the cells were treated with starvation for the indicated time .

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K. Western blot analysis of ULK1 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time.


L . Western blot analysis of p62 or LC3 levels in HeLa cells with or without STK38L knockout , the cells were treated with starvation for the indicated time . Bafilomycin A1 ( BAF A1 ) was used to inhibit the fusion between autophagosomes and lysosomes .

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L. Western blot analysis of p62 or LC3 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time. Bafilomycin A1 (BAF A1) was used to inhibit the fusion between autophagosomes and lysosomes.


A . Western blot analysis of ULK1 levels in HeLa cells stably expressing Flag - TRIM27 , the cells were transfected with either Myc - vector , Myc - STK38L , or Myc - STK38L - K119A . Then the cells were treated with starvation before lysis .

A. Western blot analysis of ULK1 levels in HeLa cells stably expressing Flag-TRIM27, the cells were transfected with either Myc-vector, Myc-STK38L, or Myc-STK38L-K119A. Then the cells were treated with starvation before lysis.


B . Phosphorylation of HA - ULK1 in HeLa STK38L knockout cells with or without expression of Flag - STK38L . HA - ULK1 was immunoprecipitated and then analyzed by western blot using anti - phosphoserine or anti - phosphothreonine , and the cell lysates were analyzed using anti - HA and anti - Myc .

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B. Phosphorylation of HA-ULK1 in HeLa STK38L knockout cells with or without expression of Flag-STK38L. HA-ULK1 was immunoprecipitated and then analyzed by western blot using anti-phosphoserine or anti-phosphothreonine, and the cell lysates were analyzed using anti-HA and anti-Myc.


C . Co - immunoprecipitation of Flag - TRIM27 with HA - ULK1 or HA - ULK1 - S495A in HEK293T cells . HA - ULK1 or HA - ULK1 S495A was immunoprecipitated using anti - HA , the immunoprecipitates and lysates were analyzed with anti - HA or anti - Flag .

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C. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 or HA-ULK1-S495A in HEK293T cells. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with anti-HA or anti-Flag.


D . Ubiquitination analysis of HA - ULK1 WT or HA - ULK1 S495A in HEK293T cells co - expressing Flag - TRIM27 and His - Ub .

D. Ubiquitination analysis of HA-ULK1 WT or HA-ULK1 S495A in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.


E . Phosphorylation of HA - ULK1 in HEK293T cells co - expressing Myc - STK38L with or without CIP treatment . HA - ULK1 was immunoprecipitated with anti - HA and analyzed by western blot using a specific antibody against p - ULK1 - S495 ( Ser495 ) .

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E. Phosphorylation of HA-ULK1 in HEK293T cells co-expressing Myc-STK38L with or without CIP treatment. HA-ULK1 was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).


F . Phosphorylation of HA - ULK1 or HA - ULK1 S495A in HEK293T cells co - expressing Myc - STK38L . HA - ULK1 or HA - ULK1 S495A was immunoprecipitated with anti - HA and analyzed by western blot using a specific antibody against p - ULK1 - S495 ( Ser495 ) .

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F. Phosphorylation of HA-ULK1 or HA-ULK1 S495A in HEK293T cells co-expressing Myc-STK38L. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).


G . In vitro kinase assay using GST - STK38L , or GST - STK38L K119A ( a kinase - inactive mutant ) and HA - ULK1 as substrate . GST - STK38L or GST - STK38L K119A was pulled down using glutathione Sepharose beads . HA - ULK1 was immunoprecipitated from HEK293T using anti - HA and treated with lambda phosphatase , the reaction product was analyzed by western blot using p - ULK1 - S495 ( Ser495 ) .

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G. In vitro kinase assay using GST-STK38L, or GST-STK38L K119A (a kinase-inactive mutant) and HA-ULK1 as substrate. GST-STK38L or GST-STK38L K119A was pulled down using glutathione Sepharose beads. HA-ULK1 was immunoprecipitated from HEK293T using anti-HA and treated with lambda phosphatase, the reaction product was analyzed by western blot using p-ULK1-S495 (Ser495).


H . Ubiquitination analysis of Myc - STK38L in HEK293T cells co - expressing Flag - TRIM27 and His - Ub .

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H. Ubiquitination analysis of Myc-STK38L in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.


I . Ubiquitination analysis of Flag - STK38L in HEK293T cells co - expressing His - Ub with or without TRIM27 knockdown , cells were cultured in fed or starved conditions as indicated .

I. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing His-Ub with or without TRIM27 knockdown, cells were cultured in fed or starved conditions as indicated.


J . Ubiquitination analysis of Flag - STK38L in HEK293T cells co - expressing Myc - TRIM27 with either His - Ub ( wild - type ) or His - Ub K6R .

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J. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing Myc-TRIM27 with either His-Ub (wild-type) or His-Ub K6R.


K . Ubiquitination analysis of Myc - STK38L WT , Myc - STK38L - K432R + K434R or Myc - STK38L - K181R + K215R + K224R + K432R + K434R ( Myc - STK38L - 5KR ) mutant in HEK293T cells co - expressing HA - TRIM27 and His - Ub .

K. Ubiquitination analysis of Myc-STK38L WT, Myc-STK38L-K432R+K434R or Myc-STK38L-K181R+K215R+K224R+K432R+K434R (Myc-STK38L-5KR) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.


L . Ubiquitination analysis of Myc - STK38L K1 - 38R mutant ( All lysines were mutated to arginines ) , or Myc - STK38L - R181 / 215 / 224 / 432 / 434K ( All lysines were mutated to arginines except the indicated five lysine residues ) mutant in HEK293T cells co - expressing HA - TRIM27 and His - Ub .

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L. Ubiquitination analysis of Myc-STK38L K1-38R mutant (All lysines were mutated to arginines), or Myc-STK38L-R181/215/224/432/434K (All lysines were mutated to arginines except the indicated five lysine residues) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.


M . Phosphorylation of HA - ULK1 S495 ( Ser495 ) in HEK293T cells co - expressing Myc - STK38L WT , or Myc - STK38L - 5KR mutant in K . The cells were cultured in fed or starved conditions as indicated and HA - ULK1 was immunoprecipitated using anti - HA , the immunoprecipitates and lysates were analyzed with a specific antibody against p - ULK1 - S495 ( Ser495 ) , anti - Myc , anti - HA , or anti - actin .

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M. Phosphorylation of HA-ULK1 S495(Ser495) in HEK293T cells co-expressing Myc-STK38L WT, or Myc-STK38L-5KR mutant in K. The cells were cultured in fed or starved conditions as indicated and HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-Myc, anti-HA, or anti-actin.


N . Phosphorylation of HA - ULK1 in HeLa cells with or without TRIM27 - knockout , cells were cultured in fed or starved conditions as indicated , HA - ULK1 was immunoprecipitated using anti - HA , the immunoprecipitates and lysates were analyzed with a specific antibody against p - ULK1 - S495 ( Ser495 ) , anti - HA , or anti - actin .

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N. Phosphorylation of HA-ULK1 in HeLa cells with or without TRIM27-knockout, cells were cultured in fed or starved conditions as indicated, HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-HA, or anti-actin.


A . Comparison of the relative mRNA levels of TRIM27 in normal breast tissues ( Normal , n = 113 ) and indicated breast tumors ( Basal ， n = 143 ； Her2 ， n = 67 ； LumA ， n = 427 ； LumB ， n = 190 ) in the TCGA database . The number of biological replicates are shown as indicated . P < 0 . 0001 ( one - way ANOVA ) .

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A. Comparison of the relative mRNA levels of TRIM27 in normal breast tissues (Normal, n=113) and indicated breast tumors (Basal，n=143；Her2，n=67；LumA，n=427；LumB，n=190) in the TCGA database. The number of biological replicates are shown as indicated. P < 0.0001 (one-way ANOVA).


B . Analysis of the correlation between ULK1 and TRIM27 expression in the CPTAC proteomics database . P = 0 . 0027 ( Pearson ' s correlation method ) .

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B. Analysis of the correlation between ULK1 and TRIM27 expression in the CPTAC proteomics database. P = 0.0027 (Pearson's correlation method).


Western blot analysis of ULK1 and TRIM27 levels from tumor tissues ( T ) , Pericarcinomatous tissues ( P ) , and their matched surrounding normal tissues ( N ) . GAPDH was used as a loading control ( C ) .

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Western blot analysis of ULK1 and TRIM27 levels from tumor tissues (T), Pericarcinomatous tissues (P), and their matched surrounding normal tissues (N). GAPDH was used as a loading control (C).


F . Representative tumors in MMTV - PyMT mice of the indicated Trim27 genotypes . Scale bar , 5mm .

F. Representative tumors in MMTV-PyMT mice of the indicated Trim27 genotypes. Scale bar, 5mm.


G . Western blot analysis of indicated proteins in Trim27 - / - PyMT or Trim27 + / - PyMT mice in primary breast tumors . Actin was used as a loading control .

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G. Western blot analysis of indicated proteins in Trim27-/- PyMT or Trim27+/- PyMT mice in primary breast tumors. Actin was used as a loading control.


H . Kaplan - Meier tumor - free survival curves of Trim27 + / - PyMT ( median = 75 d , number = 20 ) , or Trim27 - / - PyMT mice ( median = 95 d , number = 16 ) . P < 0 . 0001 ( Mantel - Cox test ) .

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H. Kaplan-Meier tumor-free survival curves of Trim27+/- PyMT (median = 75 d, number = 20), or Trim27 -/- PyMT mice (median = 95 d, number = 16). P < 0.0001 (Mantel-Cox test).


Pulmonary surface nodules in Trim27 + / - PyMT mice and Trim27 - / - PyMT mice ( I Scale bar , 1mm .

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Pulmonary surface nodules in Trim27+/- PyMT mice and Trim27-/- PyMT mice (I Scale bar, 1mm.


Pulmonary surface nodules and hematoxylin and eosin ( H & E ) staining in Trim27 + / - PyMT mice and Trim27 - / - PyMT mice J ) . Scale bar , 1mm . ( K ) . Bar chart showing quantification for the number of metastatic nodules per lung lope ( Trim27 + / - , n = 6 mice ; Trim27 - / - n = 6 mice ) , when compared at the same final timepoint . Each symbol represents a mouse ; Data are presented as mean ± s . d . from three independent experiments ; * * * P < 0 . 001 ( unpaired student ' s t - test ) .

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Pulmonary surface nodules and hematoxylin and eosin (H&E) staining in Trim27+/- PyMT mice and Trim27-/- PyMT mice J). Scale bar, 1mm. (K). Bar chart showing quantification for the number of metastatic nodules per lung lope (Trim27+/-, n=6 mice; Trim27-/- n=6 mice), when compared at the same final timepoint. Each symbol represents a mouse; Data are presented as mean ± s.d. from three independent experiments; ***P<0.001 (unpaired student's t-test).


B , BrdU incorporation assay for H460 and A549 cells . The mean ± SD represents 6 biological replicates in one experiment . Three independent experiments were performed . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

B, BrdU incorporation assay for H460 and A549 cells. The mean ± SD represents 6 biological replicates in one experiment. Three independent experiments were performed. **p < 0.01, ****p < 0.0001, unpaired two-tailed Student's t test.


D , Immunoblot showing the expression of Cleaved - PARP , Cleaved - CASP3 , CASP3 and Actin in H460 and A549 cells .

D, Immunoblot showing the expression of Cleaved-PARP, Cleaved-CASP3, CASP3 and Actin in H460 and A549 cells.


E , H460 and A549 cells were cultured in 5 % Matrigel for 10 days . Representative images of the 3D Matrigel culture are shown in the top panel , and immunofluorescence staining with anti - laminin5 and DAPI is shown in the bottom panel ( laminin5 , red ; DAPI , blue ) . Scale bars , 200 μm . The right panel shows the percentages of colonies with a diameter ≥ 50 μm and colonies with a diameter < 50 μm . The mean ± SD represents 5 visual fields from one experiment . Three independent experiments were performed . * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

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E, H460 and A549 cells were cultured in 5% Matrigel for 10 days. Representative images of the 3D Matrigel culture are shown in the top panel, and immunofluorescence staining with anti-laminin5 and DAPI is shown in the bottom panel (laminin5, red; DAPI, blue). Scale bars, 200 μm. The right panel shows the percentages of colonies with a diameter ≥ 50 μm and colonies with a diameter < 50 μm. The mean ± SD represents 5 visual fields from one experiment. Three independent experiments were performed. ****p < 0.0001, unpaired two-tailed Student's t test.


F , The relative cytoplasmic nucleosomes index was measured by detection with the cell death detection ELISA kit for H460 and A549 cells after 24 h under attached or suspended conditions . Immunoblot showing the expression of Cleaved - PARP , Cleaved - CASP3 , CASP3 and Actin in H460 and A549 cells after 24 h under attached or suspended conditions . The mean ± SD represents 4 biological replicates in one experiment . Three independent experiments were performed . ns , nonsignificant , * * p < 0 . 01 , unpaired two - tailed Student ' s t test .

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F, The relative cytoplasmic nucleosomes index was measured by detection with the cell death detection ELISA kit for H460 and A549 cells after 24 h under attached or suspended conditions. Immunoblot showing the expression of Cleaved-PARP, Cleaved-CASP3, CASP3 and Actin in H460 and A549 cells after 24 h under attached or suspended conditions. The mean ± SD represents 4 biological replicates in one experiment. Three independent experiments were performed. ns, nonsignificant, **p < 0.01, unpaired two-tailed Student's t test.


G , The indicated cells were allowed to grow in soft agar for 2 weeks , and colonies were counted . The mean ± SD represents 3 biological replicates in one experiment . Three independent experiments were performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired two - tailed Student ' s t test .

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G, The indicated cells were allowed to grow in soft agar for 2 weeks, and colonies were counted. The mean ± SD represents 3 biological replicates in one experiment. Three independent experiments were performed. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student's t test.


J , The volcano plot maps based on RNA - seq data show changes in the gene expression profile in H460 ( C / C ) cells versus H460 ( T / T ) cells .

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J, The volcano plot maps based on RNA-seq data show changes in the gene expression profile in H460 (C/C) cells versus H460 (T/T) cells.


A - C , H460 ( T / T ) , H460 ( C / C ) , A549 ( T / T ) and A549 ( C / C ) cells were subcutaneously inoculated into BALB / c nude mice ( n = 5 ) . Primary tumors were resected after 5 weeks ( A ) . The graphs show the mean tumor volumes ( B ) and weight ( C ) . Values shown are means ± SD obtained from animal experiments . * p < 0 . 05 , * * p < 0 . 01 , two - way ANOVA ( B ) and unpaired two - tailed Student ' s t test ( C ) .

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A-C, H460 (T/T), H460 (C/C), A549 (T/T) and A549 (C/C) cells were subcutaneously inoculated into BALB/c nude mice (n = 5). Primary tumors were resected after 5 weeks (A). The graphs show the mean tumor volumes (B) and weight (C). Values shown are means ± SD obtained from animal experiments. *p < 0.05, **p < 0.01, two-way ANOVA (B) and unpaired two-tailed Student's t test (C).


D , IHC staining for Ki67 in primary tumors from animals . The panels show representative images ( left ) , and the graph shows the quantification of Ki67 - positive cells in the tumor ( right ) . Scale bars , 100 μm . The mean ± SD represents 5 visual fields from each tumor tissue . * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

D, IHC staining for Ki67 in primary tumors from animals. The panels show representative images (left), and the graph shows the quantification of Ki67-positive cells in the tumor (right). Scale bars, 100 μm. The mean ± SD represents 5 visual fields from each tumor tissue. ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.


F , The scatter plot and the percentage of HLA - I - positive circulating tumor cells ( CTCs ) in the peripheral blood was analyzed by Flow Cytometry , and numerical statistics of the two groups were calculated ( n = 5 ) . Tumor - free mice were used as a negative control to determine the gating . * p < 0 . 05 , * * p < 0 . 01 , unpaired two - tailed Student ' s t test .

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F, The scatter plot and the percentage of HLA-I-positive circulating tumor cells (CTCs) in the peripheral blood was analyzed by Flow Cytometry, and numerical statistics of the two groups were calculated (n = 5). Tumor-free mice were used as a negative control to determine the gating. *p < 0.05, **p < 0.01, unpaired two-tailed Student's t test.


I , The log - rank ( Mantel - Cox ) test of overall survival of 239 Chinese lung cancer patients based on rs1663689 genotype .

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I, The log-rank (Mantel-Cox) test of overall survival of 239 Chinese lung cancer patients based on rs1663689 genotype.


F , The location diagram shows 4C enrichment around the negative control and ADGRG6 probe . Representative images of nuclei stained with DNA FISH probes for SNP rs1666389 ( green ) and negative control ( red ) or ADGRG6 ( red ) in H460 and A549 cells . The numerical points in the figure show the percentage of colocation in seven slides ( with a total of 80 nuclei ) . Arrows point to the colocation signals . Nuclei with green or red positive signal are listed in the counting range . Values shown are means ± SD . Scale bars , 5 μm . ns , nonsignificant , * p < 0 . 05 , * * p < 0 . 01 , unpaired two - tailed Student ' s t test .

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F, The location diagram shows 4C enrichment around the negative control and ADGRG6 probe. Representative images of nuclei stained with DNA FISH probes for SNP rs1666389 (green) and negative control (red) or ADGRG6 (red) in H460 and A549 cells. The numerical points in the figure show the percentage of colocation in seven slides (with a total of 80 nuclei). Arrows point to the colocation signals. Nuclei with green or red positive signal are listed in the counting range. Values shown are means ± SD. Scale bars, 5 μm. ns, nonsignificant, *p < 0.05, **p < 0.01, unpaired two-tailed Student's t test.


G , Expression of ADGRG6 at the protein levels in H460 and A549 cells .

G, Expression of ADGRG6 at the protein levels in H460 and A549 cells.


H , eQTL analysis demonstrating the correlation between the rs1663689 genotype and the expression of ADGRG6 in the clinical patient cohorts . The p value of the linear model was assessed by the Kruskal - Wallis test . Values shown are means ± SD .

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H, eQTL analysis demonstrating the correlation between the rs1663689 genotype and the expression of ADGRG6 in the clinical patient cohorts. The p value of the linear model was assessed by the Kruskal-Wallis test. Values shown are means ± SD.


A , Protein expression of ADGRG6 or Actin after ADGRG6 knockdown in T / T cells and overexpression in C / C cells .

A, Protein expression of ADGRG6 or Actin after ADGRG6 knockdown in T/T cells and overexpression in C/C cells.


Boyden chamber assay ( C ) of ADGRG6 - knockdown T / T cells and ADGRG6 - overexpressing C / C cells . Values shown are means ± SD obtained from one experiment . Scale bars , 200 μm . Three independent experiments were performed . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

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Boyden chamber assay (C) of ADGRG6-knockdown T/T cells and ADGRG6-overexpressing C/C cells. Values shown are means ± SD obtained from one experiment. Scale bars, 200 μm. Three independent experiments were performed. **p < 0.01, ****p < 0.0001, unpaired two-tailed Student's t test.


A , Immunoblot showing the expression of PKA substrate phosphorylation , PKA and Actin in the indicated cells and genotypes and the indicated cells treated with H89 .

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A, Immunoblot showing the expression of PKA substrate phosphorylation, PKA and Actin in the indicated cells and genotypes and the indicated cells treated with H89.


BrdU incorporation assay ( B ) of the indicated cells after treatment with H89 . Scale bars , 200 μm . Values shown are means ± SD obtained from one experiment . Three independent experiments were performed . ns , nonsignificant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

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BrdU incorporation assay (B) of the indicated cells after treatment with H89. Scale bars, 200 μm. Values shown are means ± SD obtained from one experiment. Three independent experiments were performed. ns, nonsignificant, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.


D , Representative images and quantification of patient - derived organoids treated with 20 μM H89 and controls . The mean ± SD represents 5 visual fields from one experiment . Scale bars , 20 μm . ns , nonsignificant , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t test .

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D, Representative images and quantification of patient-derived organoids treated with 20 μM H89 and controls. The mean ± SD represents 5 visual fields from one experiment. Scale bars, 20 μm. ns, nonsignificant, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.


A Cell cycle analysis by flow cytometry ( EdU vs . TO - PRO - 3 ) in complete medium ( ENRAD ) or in the absence of A83 - 01 ( 24 hours ) .

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A Cell cycle analysis by flow cytometry (EdU vs. TO-PRO-3) in complete medium (ENRAD) or in the absence of A83-01 (24 hours).


B Detection of proteins secreted by mouse prostate organoids in culture based on click - chemistry enrichment and mass spectrometry analysis ( n = 4 biological replicates ; data points are shown with crossing line representing mean value ) .

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B Detection of proteins secreted by mouse prostate organoids in culture based on click-chemistry enrichment and mass spectrometry analysis (n=4 biological replicates; data points are shown with crossing line representing mean value).


C IF analysis for selected markers in mouse prostate tissue and organoid sections ( scale bar = 50 μm ) .

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C IF analysis for selected markers in mouse prostate tissue and organoid sections (scale bar = 50 μm).


D Representative stereoscopic images of mouse prostate organoids cultured in the absence of A83 - 01 , with or without Follistatin ( Fst , 500 ng / mL , 6 days ) , or with or without the Activin A neutralizing antibody MAB3381 ( 1 μg / mL , 6 days ) . Scale bar = 1

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D Representative stereoscopic images of mouse prostate organoids cultured in the absence of A83-01, with or without Follistatin (Fst, 500 ng/mL, 6 days), or with or without the Activin A neutralizing antibody MAB3381 (1 μg/mL, 6 days). Scale bar = 1 mm


E Western blot ( WB ) analysis in mouse prostate organoids for selected canonical and non - canonical Tgf - β signalling mediators , and the cell cycle inhibitor p21 ( Activin A , 50 ng / mL ; Tgfb1 , 500 ng / mL ; 24 hours ) .

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E Western blot (WB) analysis in mouse prostate organoids for selected canonical and non-canonical Tgf-β signalling mediators, and the cell cycle inhibitor p21 (Activin A, 50 ng/mL; Tgfb1, 500 ng/mL; 24 hours).


G Representative stereoscopic images of mouse prostate organoids following treatment with Takinib ( Tak1 inhibitor ; 5 μM , 6 days ) , SB203580 ( p38α / β inhibitor ; 10 μM , 6 days ) or the structurally unrelated Ralimetinib ( p38α / β inhibitor ; 1 μM , 7 days ) . Scale bar = 1

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G Representative stereoscopic images of mouse prostate organoids following treatment with Takinib (Tak1 inhibitor; 5 μM, 6 days), SB203580 (p38α/β inhibitor; 10 μM, 6 days) or the structurally unrelated Ralimetinib (p38α/β inhibitor; 1 μM, 7 days). Scale bar = 1 mm


H Nuclear / Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected signalling mediators in the presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 hours ) .

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H Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected signalling mediators in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 hours).


I Gene set enrichment analysis ( GSEA ) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor ( TNF ) signalling in mouse prostate organoids cultured without A83 - 01 ( 24 hours ) versus complete medium ( ENRAD ) .

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I Gene set enrichment analysis (GSEA) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor (TNF) signalling in mouse prostate organoids cultured without A83-01 (24 hours) versus complete medium (ENRAD).


J Nuclear / Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected cell cycle regulators , in the presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 or 48 hours ) .

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J Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected cell cycle regulators, in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 or 48 hours).


A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf - β ligand inhibitor A83 - 01 ( n = 3 biological replicates per lobe - specific organoid culture ; arrow = continuous expansion ; dot = passage ; cross = culture loss ) .

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A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf-β ligand inhibitor A83-01 (n = 3 biological replicates per lobe-specific organoid culture; arrow = continuous expansion; dot = passage; cross = culture loss).


B Representative stereoscopic images of C57BL / 6J DLP # 1 and # 3 mouse prostate organoid lines during adaptation in the absence of A83 - 01 . Scale bar = 1

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B Representative stereoscopic images of C57BL/6J DLP #1 and #3 mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mm


D Gene set enrichment analysis ( GSEA ) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines ( C57 # 1 DLP , top ; C57 # 3 DLP , bottom ) adapted to grow in the absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) .

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D Gene set enrichment analysis (GSEA) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines (C57#1 DLP, top; C57#3 DLP, bottom) adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD).


E Western blot analysis in C57 # 1 and C57 # 3 DLP mouse prostate organoid lines adapted to grow in the absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) . Immunoblots are displayed for Chek1 ( ATR signalling mediator ) , Trp53 and p21 .

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E Western blot analysis in C57#1 and C57#3 DLP mouse prostate organoid lines adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD). Immunoblots are displayed for Chek1 (ATR signalling mediator), Trp53 and p21.


F Representative spectral karyotype ( SKY ) images of metaphase spreads obtained from C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread genomic instability is observed following adaptation . Scale bars = 5 µm . G - H Representative telomere FISH images - and quantification ( n = 2 biological replicates , 20 metaphases per condition ) - in C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread telomeric instability is observed following adaptation ( arrows indicate telomere doublets , One - way ANOVA , scale bars = 10 µm ) .

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F Representative spectral karyotype (SKY) images of metaphase spreads obtained from C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread genomic instability is observed following adaptation. Scale bars = 5 µm. G-H Representative telomere FISH images - and quantification (n = 2 biological replicates, 20 metaphases per condition) - in C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread telomeric instability is observed following adaptation (arrows indicate telomere doublets, One-way ANOVA, scale bars = 10 µm).


A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C . trachomatis L2 ( multiplicity of infection MOI = 1 ) in the presence or not of BP . In the indicated samples CP4d was added 2 h before infection . Cell lysates were run on SDS - PAGE , proteins were transferred to a membrane and BP incorporation was revealed with HRP - conjugated streptavidin . BP incorporation is enhanced in infected samples , and is inhibited by CP4d . The two main bands present in all samples correspond to naturally biotinylated host proteins ( Haneji & Koide , 1989 ) . After blotting the membrane was stained with Coomassie blue to control for equal loading . B - Same as in ( A ) , except that where indicated 250 μM doxycycline ( doxy , left ) or 7 μM cycloheximide ( CHX , right ) were added 24 h or 2 hpi , respectively .

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A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C. trachomatis L2 (multiplicity of infection MOI=1) in the presence or not of BP. In the indicated samples CP4d was added 2 h before infection. Cell lysates were run on SDS-PAGE, proteins were transferred to a membrane and BP incorporation was revealed with HRP-conjugated streptavidin. BP incorporation is enhanced in infected samples, and is inhibited by CP4d. The two main bands present in all samples correspond to naturally biotinylated host proteins (Haneji & Koide, 1989). After blotting the membrane was stained with Coomassie blue to control for equal loading. B - Same as in (A), except that where indicated 250 μM doxycycline (doxy, left) or 7 μM cycloheximide (CHX, right) were added 24 h or 2 hpi, respectively.


C - Whole cell lysates were prepared with TG2 + / + and TG2 - / - MEFs infected or not for 48 h with C . trachomatis L2 in the presence or not of BP , and analyzed as in ( A ) .

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C - Whole cell lysates were prepared with TG2+/+ and TG2-/- MEFs infected or not for 48 h with C. trachomatis L2 in the presence or not of BP, and analyzed as in (A).


D - Western blot with anti - TG2 antibodies on total cell lysates infected or not with C . trachomatis L2 for the indicated time . The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments , with the results of the Student ' s ratio - paired t - test . NI : not infected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 -Western blot with anti-TG2 antibodies on total cell lysates infected or not with C. trachomatis L2 for the indicated time. The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments, with the results of the Student's ratio-paired t-test. NI: not infected.


E - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . Where indicated , 40 μM CP4d was added 2 hpi . tgm2 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least four times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

O O 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 O O O O O O O O O O O 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 - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. Where indicated, 40 μM CP4d was added 2 hpi. tgm2 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least four times. P-values of Student's ratio-paired t-test <0.05 are shown.


F - Cells were treated for 18 h with the indicated concentration of human recombinant IL - 6 before measuring TG2 transcription relative to actin like in ( E ) . The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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

F - Cells were treated for 18 h with the indicated concentration of human recombinant IL-6 before measuring TG2 transcription relative to actin like in (E). The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.


Cells were left uninfected or infected with C . trachomatis L2 in the presence of the indicated concentration of anti - IL - 6 receptor antibodies . Forty - eight h later TG2 transcription relative to actin was measured like in ( E ) . The data are presented as relative mRNA levels compared to uninfected / untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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

Cells were left uninfected or infected with C. trachomatis L2 in the presence of the indicated concentration of anti-IL-6 receptor antibodies. Forty-eight h later TG2 transcription relative to actin was measured like in (E). The data are presented as relative mRNA levels compared to uninfected/untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.


A - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were disrupted and bacterial titers ( IFU = inclusion forming unit ) were determined by re - infecting fresh HeLa cells as described in the methods . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

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 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 pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were disrupted and bacterial titers (IFU=inclusion forming unit) were determined by re-infecting fresh HeLa cells as described in the methods. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


B - HeLa cells were transfected with control siRNA or two siRNAs against TG2 . Two days later , the efficiency of the silencing was assessed by western blot using anti - TG2 antibodies and anti - actin antibodies as loading control ( bottom ) . Duplicate wells were infected with L2incDGFP and progeny was analyzed as in ( A ) ( top ) . The mean ± SD of four independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells were transfected with control siRNA or two siRNAs against TG2. Two days later, the efficiency of the silencing was assessed by western blot using anti-TG2 antibodies and anti-actin antibodies as loading control (bottom). Duplicate wells were infected with L2incDGFP and progeny was analyzed as in (A) (top). The mean ± SD of four independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


C - Same as in ( A ) except that C . trachomatis serovar L2 ( left ) or D ( right ) were grown in primary cells isolated from fallopian tubes . For serovar D , IFU were determined 48 hpi . The mean ± SD of four to five independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Same as in (A) except that C. trachomatis serovar L2 (left) or D (right) were grown in primary cells isolated from fallopian tubes. For serovar D, IFU were determined 48 hpi. The mean ± SD of four to five independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


D - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( left ) and the mean fluorescence of the infected population ( right ) ± SD are shown for three independent experiments . P - values of Student ' s paired t - test are indicated when < 0 . 05 . A representative field for each condition is shown , scale bar = 10 μm .

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 O O O O O O O O O O O O O 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 pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were fixed and analyzed by flow cytometry. The percentage of infected cells (left) and the mean fluorescence of the infected population (right) ± SD are shown for three independent experiments. P-values of Student's paired t-test are indicated when <0.05. A representative field for each condition is shown, scale bar = 10 μm.


E - Primary epithelial cells isolated from fallopian tubes were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with C . trachomatis serovar L2 ( left ) or D ( right ) . Twenty - four hours later the cells were fixed , bacteria were stained using FITC - labeled anti - Chlamydia - LPS antibodies , and the mean size of the inclusions manually determined using ImageJ , on twenty inclusions per experiment . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

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 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

E - Primary epithelial cells isolated from fallopian tubes were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with C. trachomatis serovar L2 (left) or D (right). Twenty-four hours later the cells were fixed, bacteria were stained using FITC-labeled anti-Chlamydia-LPS antibodies, and the mean size of the inclusions manually determined using ImageJ, on twenty inclusions per experiment. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


F - Mice were infected intravaginally with 105 IFU of C . muridarum . Twenty - five days later the mice were sacrificed and the upper genital tract , from the uterine horn to the oviduct , was collected . The right part was used for bacterial burden assessment ( top left ) . The left part was rinsed with PBS and observed with a binocular magnifier ( right ) to determine the hydrosalpinx score ( bottom left ) . Each dot represents one mouse , the mean ± SD is shown . P - values of Mann - Whitney test are indicated when < 0 . 05 .

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

F - Mice were infected intravaginally with 105 IFU of C. muridarum. Twenty-five days later the mice were sacrificed and the upper genital tract, from the uterine horn to the oviduct, was collected. The right part was used for bacterial burden assessment (top left). The left part was rinsed with PBS and observed with a binocular magnifier (right) to determine the hydrosalpinx score (bottom left). Each dot represents one mouse, the mean ± SD is shown. P-values of Mann-Whitney test are indicated when <0.05.


A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria ( MOI = 0 . 2 ) . Cells were disrupted 30 h later and the bacterial titer determined by re - infecting fresh wild type cells . The mean ± SD of three independent experiments are shown .

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

A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria (MOI = 0.2). Cells were disrupted 30 h later and the bacterial titer determined by re-infecting fresh wild type cells. The mean ± SD of three independent experiments are shown.


B - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 48 h . Where indicated 40 μM CP4d was added 2 hpi . GLUT - 1 and GLUT - 3 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated four times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

O 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 O O O O O O O O O O O O O O O O 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 - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 48 h. Where indicated 40 μM CP4d was added 2 hpi. GLUT-1 and GLUT-3 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated four times. P-values of Student's ratio-paired t-test are indicated when <0.05.


C - Relationship between TGM2 and GLUT - 1 ( top ) and GLUT - 3 ( bottom ) expression across 265 HGSOCs . Expression comparisons were performed using Spearman ' s rank correlation test .

O O O O O O O O O O O O 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 -Relationship between TGM2 and GLUT-1 (top) and GLUT-3 (bottom) expression across 265 HGSOCs. Expression comparisons were performed using Spearman's rank correlation test.


D - HeLa cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . HIF - 1α transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative

O O O O O O O O O O O O O O O O O O O O O O O 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 infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. HIF-1α transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative


E - HeLa cells were transfected with control siRNA or two siRNAs against HIF - 1α . Two days later , cells were infected with C . trachomatis L2 ( MOI = 1 ) for 48 h . The indicated transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells were transfected with control siRNA or two siRNAs against HIF-1α. Two days later, cells were infected with C. trachomatis L2 (MOI=1) for 48 h. The indicated transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.


F - TG2 - / - MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C . trachomatis L2 ( MOI = 1 ) . Mouse GLUT - 1 transcripts were measured by real - time RT - PCR and normalized to mouse actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

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

F - TG2-/- MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C. trachomatis L2 (MOI=1). Mouse GLUT-1 transcripts were measured by real-time RT-PCR and normalized to mouse actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.


A - HeLa cells were infected with C . trachomatis ( MOI = 1 ) and 40 µM CP4d was added or not 2 hpi . After 24 h 0 . 5 mM BP was added and cells were lysed at 48 hpi . Lysates were precipitated with streptavidin - coated beads . After separation with SDS - PAGE , proteins were transferred to a membrane and blotted with anti - GFPT antibody followed with HRP - conjugated secondary antibody .

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

A - HeLa cells were infected with C. trachomatis (MOI = 1) and 40 µM CP4d was added or not 2 hpi. After 24 h 0.5 mM BP was added and cells were lysed at 48 hpi. Lysates were precipitated with streptavidin-coated beads. After separation with SDS-PAGE, proteins were transferred to a membrane and blotted with anti-GFPT antibody followed with HRP-conjugated secondary antibody.