C , D H1975 and A549 cells were exposed to PM2 . 5 at 50 μg / ml for different lengths of time , and the cell lysates of treated cells were assessed for phosphorylated EGFR ( pEGFR ) and EGFR by Western blotting . β - actin served as the loading control .

C, D H1975 and A549 cells were exposed to PM2.5 at 50 μg/ml for different lengths of time, and the cell lysates of treated cells were assessed for phosphorylated EGFR (pEGFR) and EGFR by Western blotting. β-actin served as the loading control.


E , F H1975 and A549 cells were treated with PM2 . 5 at 50 μg / ml for 90 days . The anchorage - independent growth was assessed by a soft agar colony formation assay . The number of colonies was scored , and the data are presented as the relative colony formation ability .

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E, F H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days. The anchorage-independent growth was assessed by a soft agar colony formation assay. The number of colonies was scored, and the data are presented as the relative colony formation ability.


H1975 cells were exposed to 50 μg / ml PM2 . 5 for 90 days . Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse ( n = six per group ) . The tumor volume and excised tumor weight were measured . The results shown in ( A ) are presented as the means ± SDs of six mice . * p < 0 . 05 and * * p < 0 . 01 , compared with untreated group . ( A ) p values were determined by two - way repeated measures ANOVA with pairwise comparison of post - hoc analysis with Benjamini - Hochberg ( BH ) correction .

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H1975 cells were exposed to 50 μg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = six per group). The tumor volume and excised tumor weight were measured. The results shown in (A) are presented as the means ± SDs of six mice. *p < 0.05 and **p < 0.01, compared with untreated group. (A) p values were determined by two-way repeated measures ANOVA with pairwise comparison of post-hoc analysis with Benjamini-Hochberg (BH) correction.


H1975 cells were exposed to 50 μg / ml PM2 . 5 for 90 days . Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse ( n = six per group ) . C . IHC staining of excised tumors for Ki - 67 , IL18 , and TMPRSS2 is shown in ( C ) . Scale bars , 50 μm .

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H1975 cells were exposed to 50 μg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = six per group). C. IHC staining of excised tumors for Ki-67, IL18, and TMPRSS2 is shown in (C). Scale bars, 50 μm.


H1975 cells were treated with 50 μg / ml PM2 . 5 for 90 days , and the expression levels of TMPRSS2 mRNA were determined by real - time RT ‒ PCR ( A ) respectively . ( A ) p values were determined by one - sample t - test .

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H1975 cells were treated with 50 μg/ml PM2.5 for 90 days, and the expression levels of TMPRSS2 mRNA were determined by real-time RT‒PCR (A) respectively. (A) p values were determined by one-sample t-test.


C The proteins from whole cell lysates ( WCL ) and nuclear fractions ( CN ) were analyzed for AhR , Lamin B ( nuclei marker ) , and Vimentin ( cytoplasm marker ) by Western blotting . The data shown represent the mean ± SD of three independent experiments . * p < 0 . 05 and * * p < 0 . 01 , and * * * p < 0 . 001 , compared with untreated cells . # p < 0 . 05 , compared with PM2 . 5 treated cells .

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C The proteins from whole cell lysates (WCL) and nuclear fractions (CN) were analyzed for AhR, Lamin B (nuclei marker), and Vimentin (cytoplasm marker) by Western blotting. The data shown represent the mean ± SD of three independent experiments. *p < 0.05 and **p< 0.01, and ***p < 0.001, compared with untreated cells. #p < 0.05, compared with PM2.5 treated cells.


E Western blot analysis of TMPRSS2 expression in cells treated with CH223191 at 10 μM for 48 h . The relative expression level of TMPRSS2 was quantified by normalized with β - actin and are shown at the right panel . : The data shown represent the mean ± SD of three independent experiments . * p < 0 . 05 and * * p < 0 . 01 , and * * * p < 0 . 001 , compared with untreated cells . # p < 0 . 05 , compared with PM2 . 5 treated cells .

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E Western blot analysis of TMPRSS2 expression in cells treated with CH223191 at 10 μM for 48 h. The relative expression level of TMPRSS2 was quantified by normalized with β-actin and are shown at the right panel. : The data shown represent the mean ± SD of three independent experiments. *p < 0.05 and **p< 0.01, and ***p < 0.001, compared with untreated cells. #p < 0.05, compared with PM2.5 treated cells.


A Expression of TMPRSS2 was examined by Western blots of four lung cancer cell lines ( H460 , A546 , H1299 , and H1975 ) and two normal fibroblasts ( MRC5 and IMR90 ) . The results shown in ( A are from one of three similar experiments . β - actin was used as the loading control .

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A Expression of TMPRSS2 was examined by Western blots of four lung cancer cell lines (H460, A546, H1299, and H1975) and two normal fibroblasts (MRC5 and IMR90). The results shown in (A are from one of three similar experiments. β-actin was used as the loading control.


H1975 cells were infected with sh - TMPRSS2 ( sh - TMPRSS2 - 1 and sh - TMPRSS2 - 2 ) or empty vector ( Vector ) . The stable clones of TMPRSS2 knockdown cells were analyzed and their ability to perform anchorage - independent growth in soft agar ( C ) . The data shown in ( C ) represent the means ± SDs from three independent experiments . ( C ) p values were determined by one - sample t - test .

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H1975 cells were infected with sh-TMPRSS2 (sh-TMPRSS2-1 and sh-TMPRSS2-2) or empty vector (Vector). The stable clones of TMPRSS2 knockdown cells were analyzed and their ability to perform anchorage-independent growth in soft agar (C). The data shown in (C) represent the means ± SDs from three independent experiments. (C) p values were determined by one-sample t-test.


A - C H1299 cells were transfected with TMPRSS2 - HA or the empty vector ( Vector ) . After 48 h , the transfected cells were assayed for the expression of TMPRSS2 ( A ) and for the induction of IL18 by qRT ‒ PCR ( B ) and Western blotting ( C ) . The results shown in ( A , C are from one of three similar experiments . β - actin was used as the loading control .

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A-C H1299 cells were transfected with TMPRSS2-HA or the empty vector (Vector). After 48 h, the transfected cells were assayed for the expression of TMPRSS2 (A) and for the induction of IL18 by qRT‒PCR (B) and Western blotting (C). The results shown in (A, C are from one of three similar experiments. β-actin was used as the loading control.


The expression level of IL18 was examined in A549 cells exposed to PM2 . 5 at 50 μg / ml for 90 days Western blotting The results shown in H ) are from one of three similar experiments . β - actin was used as the loading control . The data shown in H ) was normalized to β - actin from three independent experiments .

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The expression level of IL18 was examined in A549 cells exposed to PM2.5 at 50 μg/ml for 90 days Western blotting The results shown in H) are from one of three similar experiments. β-actin was used as the loading control. The data shown in H) was normalized to β-actin from three independent experiments.


A IHC staining of TMPRSS2 , IL18 , and nuclear AhR in a representative normal lung tissue section . Scale bars , 2 . 5 mm ( low magnification ) and 100 μm ( high magnification ) .

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A IHC staining of TMPRSS2, IL18, and nuclear AhR in a representative normal lung tissue section. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).


B IHC staining of TMPRSS2 , IL18 , and nuclear AhR in lung cancer tissues that displayed high or low expression of TMPRSS2 . Scale bars , 2 . 5 mm ( low magnification ) and 100 μm ( high magnification ) .

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B IHC staining of TMPRSS2, IL18, and nuclear AhR in lung cancer tissues that displayed high or low expression of TMPRSS2. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).


B Average seizure score of KA - treated WT and Fxr2 KO mice over time from t = 0 ( injection time ) to 2 h . * p < 0 . 05 compared to WT mice ( two - way Repeated Measures ANOVA ; interaction effect of time and genotype ; F1 , 666 = 10 . 05 , p = 0 . 0016 ) . KA - treated WT mice ( n = 16 ) , KA - treated Fxr2 KO mice ( n = 12 ) . Data are presented as mean ± Standard Error of the Mean ( SEM ) . D Average seizure score of pilocarpine - treated WT and Fxr2 KO mice over time from t = 0 ( injection time ) to 2 h ( two - way Repeated Measures ANOVA ) . Pilocarpine - treated WT mice ( n = 6 ) , pilocarpine - treated Fxr2 KO mice ( n = 6 ) . Data are presented as mean ± SEM .

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B Average seizure score of KA-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h. * p < 0.05 compared to WT mice (two-way Repeated Measures ANOVA; interaction effect of time and genotype; F1,666 = 10.05, p = 0.0016). KA-treated WT mice (n=16), KA-treated Fxr2 KO mice (n=12). Data are presented as mean ± Standard Error of the Mean (SEM). D Average seizure score of pilocarpine-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h (two-way Repeated Measures ANOVA). Pilocarpine-treated WT mice (n=6), pilocarpine-treated Fxr2 KO mice (n=6). Data are presented as mean ± SEM.


B PSD - 95 mRNA had lower read counts in Fxr2 KO samples ( n = 3 ) compared to WT samples ( n = 3 ) ( + adjusted p < 0 . 1 , two - tailed Student ' s t - test ) . Data are presented as mean ± SEM .

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B PSD-95 mRNA had lower read counts in Fxr2 KO samples (n=3) compared to WT samples (n=3) (+ adjusted p < 0.1, two-tailed Student's t-test). Data are presented as mean ± SEM.


A Representative Western blot and quantification of GluK5 , PSD - 95 , GluN2B and mGluR5 proteins . Endogenous GluK5 , PSD - 95 , GluN2B and mGluR5 in Fxr2 KO mice ( n = 9 ) compared to WT mice ( n = 11 ) . * p < 0 . 05 , * * p < 0 . 01 , significant difference between WT and Fxr2 KO ( two - tailed Student ' s t - test : GluK5 : t18 = 0 . 87 , p = 0 . 394 ; PSD - 95 : t18 = 2 . 29 , p < 0 . 05 ; GluN2B : t18 = 3 . 09 , p < 0 . 01 ; mGluR5 : t18 = 3 . 74 , p < 0 . 01 ) . Quantified bands are highlighted with an asterisk or squared bracket . Data are presented as mean ± SEM .

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A Representative Western blot and quantification of GluK5, PSD-95, GluN2B and mGluR5 proteins. Endogenous GluK5, PSD-95, GluN2B and mGluR5 in Fxr2 KO mice (n=9) compared to WT mice (n=11). * p < 0.05, ** p < 0.01, significant difference between WT and Fxr2 KO (two-tailed Student's t-test: GluK5: t18 = 0.87, p = 0.394; PSD-95: t18 = 2.29, p < 0.05; GluN2B: t18 = 3.09, p < 0.01; mGluR5: t18 = 3.74, p < 0.01). Quantified bands are highlighted with an asterisk or squared bracket. Data are presented as mean ± SEM.


A Bar plot showing phospho - ERK1 / 2 over total ERK1 / 2 levels in vehicle - and KA - treated WT ( closed and open black circles , vehicle - treated WT n = 6 ; KA - treated WT n = 6 ) and Fxr2 KO mice ( closed and open magenta circles , vehicle - treated Fxr2 KO n = 5 ; KA - treated Fxr2 KO n = 6 ) . * * * p < 0 . 001 comparison between vehicle - and KA - treated WT ( two - way ANOVA : interaction effect between genotype and treatment : F1 , 19 = 24 . 66 , p < 0 . 001 ) . Data are presented as mean ± SEM . B Phospho - ERK1 / 2 levels in vehicle - and pilocarpine - treated WT ( n = 4 - 6 ) and Fxr2 KO mice ( n = 4 - 6 ) . Both genotypes responded with long - lasting seizures upon pilocarpine administration * p < 0 . 05 ( two - way ANOVA : main effect for treatment : F1 , 16 = 10 . 98 , p < 0 . 01 ) . Data are presented as mean ± SEM .

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A Bar plot showing phospho-ERK1/2 over total ERK1/2 levels in vehicle- and KA-treated WT (closed and open black circles, vehicle-treated WT n=6; KA-treated WT n=6) and Fxr2 KO mice (closed and open magenta circles, vehicle-treated Fxr2 KO n=5; KA-treated Fxr2 KO n=6). *** p < 0.001 comparison between vehicle- and KA-treated WT (two-way ANOVA: interaction effect between genotype and treatment: F1,19 = 24.66, p < 0.001). Data are presented as mean ± SEM. B Phospho-ERK1/2 levels in vehicle- and pilocarpine-treated WT (n=4-6) and Fxr2 KO mice (n=4-6). Both genotypes responded with long-lasting seizures upon pilocarpine administration * p < 0.05 (two-way ANOVA: main effect for treatment: F1,16 = 10.98, p < 0.01). Data are presented as mean ± SEM.


B Recombinant MAVS - Region III - dimer ( lanes 1 - 4 ) , trimer ( lanes 5 - 8 ) or tetramer ( lanes 9 - 12 ) were subjected to IRF3 dimerization assay respectively in vitro . Increasing doses of recombinant MAVS - Region III were used and shown in Fig EV1C . Immunoblotting analysis was performed following Native - PAGE for the reaction mixture .

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B Recombinant MAVS-Region III-dimer (lanes 1-4), trimer (lanes 5-8) or tetramer (lanes 9-12) were subjected to IRF3 dimerization assay respectively in vitro. Increasing doses of recombinant MAVS-Region III were used and shown in Fig EV1C. Immunoblotting analysis was performed following Native-PAGE for the reaction mixture.


C pcDNA3 - based constructs expressing MAVS - Region III - dimer , trimer and tetramer were transfected into HEK293T cells respectively . ISG54 induction were measured thirty - six hours after transfection by quantitative PCR ( qPCR ) .

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C pcDNA3-based constructs expressing MAVS-Region III-dimer, trimer and tetramer were transfected into HEK293T cells respectively. ISG54 induction were measured thirty-six hours after transfection by quantitative PCR (qPCR).


D Recombinant MAVS - Region III - dimer or tetramer were immunoprecipitated with anti - FLAG M2 beads with or without a preincubation with S100 from HEK293T cells following an experimental procedure outlined in Fig EV1G . The IP products were resolved in SDS - PAGE and a specific protein band pulled - down with MAVS - Region III - tetramer was indicated by an arrow . TRAF3IP3 was identified in the specific band by mass spectrometry .

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D Recombinant MAVS-Region III-dimer or tetramer were immunoprecipitated with anti-FLAG M2 beads with or without a preincubation with S100 from HEK293T cells following an experimental procedure outlined in Fig EV1G. The IP products were resolved in SDS-PAGE and a specific protein band pulled-down with MAVS-Region III-tetramer was indicated by an arrow. TRAF3IP3 was identified in the specific band by mass spectrometry.


E Protein samples as described in ( D ) were subjected to immunoblotting analysis . The TRAF3IP3 band was indicated by an arrow and the asterisk indicated nonspecific

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E Protein samples as described in (D) were subjected to immunoblotting analysis. The TRAF3IP3 band was indicated by an arrow and the asterisk indicated nonspecific bands


F pcDNA3 - FLAG - TRAF3IP3 ( full - length and truncated forms ) and pcDNA3 - HA - MAVS were transfected into HEK293T cells , which were harvested thirty - six hours post transfection . Co - IP experiments were performed with anti - HA antibody , followed by immunoblotting .

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F pcDNA3-FLAG-TRAF3IP3 (full-length and truncated forms) and pcDNA3-HA-MAVS were transfected into HEK293T cells, which were harvested thirty-six hours post transfection. Co-IP experiments were performed with anti-HA antibody, followed by immunoblotting.


G Human cells , including THP1 , HeLa , HEK293 , HEK293T as well as Traf3ip3 - / - HEK293T , were lysed and subjected to immunoblotting to examine TRAF3IP3 expression .

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G Human cells, including THP1, HeLa, HEK293, HEK293T as well as Traf3ip3-/- HEK293T, were lysed and subjected to immunoblotting to examine TRAF3IP3 expression.


A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency . A pair of scrambled RNA oligoes was transfected and signified as con .

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A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency. A pair of scrambled RNA oligoes was transfected and signified as con.


B Three pairs of si - TRAF3IP3 together with IFNβ - luciferase reporter were transfected into HEK 293T cells . Twenty - four hours after transfection , the cells were infected with ( SeV ) or without ( mock ) Sendai virus , which were harvested six hours post infection to measure the luciferase activity .

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B Three pairs of si-TRAF3IP3 together with IFNβ-luciferase reporter were transfected into HEK 293T cells. Twenty-four hours after transfection, the cells were infected with (SeV) or without (mock) Sendai virus, which were harvested six hours post infection to measure the luciferase activity.


C Si - TRAF3IP3 ( oligo # 1 ) were transfected into HEK 293 cells , which were then treated with mock , VSV , SeV , poly ( I : C ) or HSV - 1 . After various treatments , IFNB inductions were measured by qPCR .

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C Si-TRAF3IP3 (oligo #1) were transfected into HEK 293 cells, which were then treated with mock, VSV, SeV, poly(I:C) or HSV-1. After various treatments, IFNB inductions were measured by qPCR.


D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3 - / - HEK293T cells .

D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3-/- HEK293T cells.


E Wild type and Traf3ip3 - / - HEK293T cells were treated with or without VSV , SeV or poly ( I : C ) as indicated for twelve hours . IFNB induction was measured by qPCR .

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E Wild type and Traf3ip3-/- HEK293T cells were treated with or without VSV, SeV or poly (I:C) as indicated for twelve hours. IFNB induction was measured by qPCR.


F Wild type and Traf3ip3 - / - HEK293T cells were infected with GFP - VSV . Fluorescent images were taken to examine GFP - VSV proliferation at eight hours post infection ( left ) . Scale bar represents 10 micrometers . GFP - VSV titers were quantitated by plaque assay ( right ) .

F Wild type and Traf3ip3-/- HEK293T cells were infected with GFP-VSV. Fluorescent images were taken to examine GFP-VSV proliferation at eight hours post infection (left). Scale bar represents 10 micrometers. GFP-VSV titers were quantitated by plaque assay (right).


G Various expression constructs were transfected into WT and Traf3ip3 - / - HEK293T cells . Thirty - six hours after transfection , IFNB induction was measured by qPCR .

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G Various expression constructs were transfected into WT and Traf3ip3-/- HEK293T cells. Thirty-six hours after transfection, IFNB induction was measured by qPCR.


H Mavs - / - HEK293T cells were transfected with empty vector , pcDNA - FLAG - MAVS , pcDNA - FLAG - MAVS - ( Region III only ) , or pcDNA - FLAG - MAVS - ( ΔRegion III ) , together with pcDNA3 - FLAG - TRAF3IP3 or not as indicated . Twenty - four hours after transfection , cells were infected with SeV for twelve hours . IFNB induction was measured by qPCR .

H Mavs-/- HEK293T cells were transfected with empty vector, pcDNA-FLAG-MAVS, pcDNA-FLAG-MAVS-(Region III only), or pcDNA-FLAG-MAVS-(ΔRegion III), together with pcDNA3-FLAG-TRAF3IP3 or not as indicated. Twenty-four hours after transfection, cells were infected with SeV for twelve hours. IFNB induction was measured by qPCR.


A The experiment was performed as described in Fig 2C . Instead of IFNB , ISG54 and IL6 inductions were measured by qPCR .

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A The experiment was performed as described in Fig 2C. Instead of IFNB, ISG54 and IL6 inductions were measured by qPCR.


B HEK293 cells were transfected with si - TRAF3IP3 oligos and then treated with various stimuli . Following subcellular fractionation , S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting . The whole cell lysates were also subjected to SDS - PAGE for anti - IRF3 and tubulin immunoblotting .

B HEK293 cells were transfected with si-TRAF3IP3 oligos and then treated with various stimuli. Following subcellular fractionation, S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting. The whole cell lysates were also subjected to SDS-PAGE for anti- IRF3 and tubulin immunoblotting.


C The experiment was performed as described in Fig 2E . ISG54 and IL6 induction was measured by qPCR .

C The experiment was performed as described in Fig 2E. ISG54 and IL6 induction was measured by qPCR.


D Wild type and Traf3ip3 - / - HEK293T cells were infected with or without VSV . At indicated time points , Cells were collected and analyzed by immunoblotting . Following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , and P5 fractions contained mitochondria were subjected to SDD - AGE to examine MAVS aggregation . The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control .

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D Wild type and Traf3ip3-/- HEK293T cells were infected with or without VSV. At indicated time points, Cells were collected and analyzed by immunoblotting. Following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation. The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control.


A pcDNA - FLAG - TRADD and pcDNA - FLAG - TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively . ISG54 and IL6 induction were measured thirty - six hours post - transfection by qPCR . Expression levels were shown in Fig EV4B . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 NS indicates no statistically significant difference .

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A pcDNA-FLAG-TRADD and pcDNA-FLAG-TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively. ISG54 and IL6 induction were measured thirty-six hours post-transfection by qPCR. Expression levels were shown in Fig EV4B. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05, **P<0.01 and ***P<0.001 NS indicates no statistically significant difference.


B HEK293T cells were transfected with pcDNA3 - HA - TRAF3IP3 , in combination with pcDNA3 - FLAG - RIG - I , MAVS , TBK1 , IRF3 , TRAF2 , TRAF3 , TRAF5 , or TRAF6 as indicated . Thirty - six hours after transfection , cells were collected and anti - HA immunoprecipitations were performed . IP products were subjected to immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293T cells were transfected with pcDNA3-HA-TRAF3IP3, in combination with pcDNA3-FLAG- RIG-I, MAVS, TBK1, IRF3, TRAF2, TRAF3, TRAF5, or TRAF6 as indicated. Thirty-six hours after transfection, cells were collected and anti-HA immunoprecipitations were performed. IP products were subjected to immunoblotting.


C shRNA targeting TRAF3IP3 ( sh - TRAF3IP3 ) was transduced into Mavs - / - HEK293T cells . Twenty - four hours after transduction , cells were treated with puromycin ( 2 μg / mL ) for forty - eight hours and then transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours after transfection , cells were infected with or without VSV for twelve hours . Cells were then collected and subjected to immunoprecipitation assay and immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 shRNA targeting TRAF3IP3 (sh-TRAF3IP3) was transduced into Mavs-/- HEK293T cells. Twenty-four hours after transduction, cells were treated with puromycin (2 μg/mL) for forty-eight hours and then transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours after transfection, cells were infected with or without VSV for twelve hours. Cells were then collected and subjected to immunoprecipitation assay and immunoblotting.


D Wild type and Traf3ip3 - / - HEK293T cells were transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours post - transfection , cells were infected with or without VSV . Twelve hours post - infection , cells were collected and subjected to immunoprecipitation assay and immunoblotting .

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D Wild type and Traf3ip3-/- HEK293T cells were transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours post-transfection, cells were infected with or without VSV. Twelve hours post-infection, cells were collected and subjected to immunoprecipitation assay and immunoblotting.


A Subcellular fractionation was performed for HEK293T cells infected with or without SeV to get three fractions , i . e . , cytoplasm , membrane and nuclei extracts , which were then subjected to Immunoblotting analysis as indicated .

A Subcellular fractionation was performed for HEK293T cells infected with or without SeV to get three fractions, i.e., cytoplasm, membrane and nuclei extracts, which were then subjected to Immunoblotting analysis as indicated.


B Membranous fraction obtained in Fig 5A were further separated into mitochondria , Golgi and ER extracts as described in methods , followed by immunoblotting analysis .

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B Membranous fraction obtained in Fig 5A were further separated into mitochondria, Golgi and ER extracts as described in methods, followed by immunoblotting analysis.


C - E Immunofluorescent microscopic imaging for TRAF3IP3 . HeLa cells were transfected with pcDNA3 - FLAG - TRAF3IP3 . Twenty - four hours after transfection , cells were infected with or without SeV for twelve hours . Anti - FLAG M2 ( FITC ) was used for immunofluorescence staining of FLAG - TRAF3IP3 . Nuclei were stained with DAPI . Mitochondria was stained with Mitotracker Red ( C ) . Calnexin ( an ER protein ) was stained for ER ( D ) . GM130 ( a Golgi protein ) was stained for Golgi ( E ) . Scale bar represents 5 micrometers . F Quantitative analysis of yellow color as shown in Fig 5C , D and E , indicating TRAF3IP3 localization on various organelles with or without virus infection . The quantification was performed with ImageJ . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * * * P < 0 . 001 . NS indicates no statistically significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Immunofluorescent microscopic imaging for TRAF3IP3. HeLa cells were transfected with pcDNA3-FLAG-TRAF3IP3. Twenty-four hours after transfection, cells were infected with or without SeV for twelve hours. Anti-FLAG M2 (FITC) was used for immunofluorescence staining of FLAG-TRAF3IP3. Nuclei were stained with DAPI. Mitochondria was stained with Mitotracker Red (C). Calnexin (an ER protein) was stained for ER (D). GM130 (a Golgi protein) was stained for Golgi (E). Scale bar represents 5 micrometers. F Quantitative analysis of yellow color as shown in Fig 5C, D and E, indicating TRAF3IP3 localization on various organelles with or without virus infection. The quantification was performed with ImageJ. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. ***P<0.001. NS indicates no statistically significant difference.


BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were treated with or without VSV , SeV , poly ( I : C ) or HSV - 1 respectively as indicated for six hours . Ifnb ( A ) , Ifna4 ( B ) , Il6 induction was measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference .

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 I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Ifnb (A), Ifna4 (B), Il6 induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.


BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were treated with or without VSV , SeV , poly ( I : C ) or HSV - 1 respectively as indicated for six hours . Il6 ( C ) , Isg54 ( D ) , and Cxcl10 ( E ) induction was measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference .

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 I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Il6 (C), Isg54 (D), and Cxcl10 (E) induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.


F BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were infected with or without VSV for six hours . Cells were then collected and following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , and P5 fractions contained mitochondria were subjected to SDD - AGE to examine MAVS aggregation .

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F BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were infected with or without VSV for six hours. Cells were then collected and following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation.


Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The spleens were collected 4 hours or 8 hours after infection and Ifna4 ( A ) , Ifnb ( B ) , Isg54 ( C ) and Il6 ( D ) inductions were measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference .

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

Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and Ifna4 (A), Ifnb (B), Isg54 (C) and Il6 (D) inductions were measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.


Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR ( E ) . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference .

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

Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR (E). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.


Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA ( F ) . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference .

Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA (F). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.


G Wild type , Traf3ip3 - / - and Mavs - / - mice ( n = 6 each ) were injected with VSV via tail vein injection at 5 × 107 pfu per mouse , and the survival rates were monitored for 15 days . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 .

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G Wild type, Traf3ip3-/- and Mavs-/- mice (n=6 each) were injected with VSV via tail vein injection at 5×107 pfu per mouse, and the survival rates were monitored for 15 days. P values were determined by unpaired two-tailed Student's t-test. *P<0.05


( F ) Malate dehydrogenase ( MDH ; 0 . 2 µM ) was incubated at 40 ° C in the absence ( black ) or presence of 0 . 5 µM WT HSP27 ( red ) or 0 . 5 µM P182L HSP27 ( blue ) . ( G ) Insulin ( 40 µM ) was incubated at 40 ° C in the absence ( black ) or presence of 20 µM WT HSP27 ( red ) or 20 µM P182L HSP27 ( blue ) . The y - axes depict the absorbance at 340 nm due to the formation of large aggregates . The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation .

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(F) Malate dehydrogenase (MDH; 0.2 µM) was incubated at 40 °C in the absence (black) or presence of 0.5 µM WT HSP27 (red) or 0.5 µM P182L HSP27 (blue). (G) Insulin (40 µM) was incubated at 40 °C in the absence (black) or presence of 20 µM WT HSP27 (red) or 20 µM P182L HSP27 (blue). The y-axes depict the absorbance at 340 nm due to the formation of large aggregates. The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation.


( A ) SEC - MALS data from WT HSP27 and the P182L variant , with cHSP27 included for comparison . All samples were injected at 40 µM concentration ( monomer ) . The respective average molecular masses are 14 MDa , 470 kDa , and 18 kDa for P182L , WT HSP27 , and cHSP27 . The normalized UV signals are plotted on the left y - axis as solid lines and the molecular masses are plotted on the right y - axis as circles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SEC-MALS data from WT HSP27 and the P182L variant, with cHSP27 included for comparison. All samples were injected at 40 µM concentration (monomer). The respective average molecular masses are 14 MDa, 470 kDa, and 18 kDa for P182L, WT HSP27, and cHSP27. The normalized UV signals are plotted on the left y-axis as solid lines and the molecular masses are plotted on the right y-axis as circles.


( B ) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10 % to 80 % sucrose gradient . Western blot against anti - V5 is 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

(B) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10% to 80% sucrose gradient. Western blot against anti-V5 is shown.


( C ) Expression of V5 - epitope - tagged HSP27 ( WT or P182L mutant ) in HSP27 knock - out HeLa cells and immunostaining of HSP27 . The scale bars correspond to the non - expanded dimensions , derived by dividing by the sample expansion factor of 4 . 3 . ( D ) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples ( n = 30 ) . The error bar is the standard deviation for the different cytoplasmic regions . ( E ) Distribution of the intensity of individual spots ( n = 3342 ) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots .

(C) Expression of V5-epitope-tagged HSP27 (WT or P182L mutant) in HSP27 knock-out HeLa cells and immunostaining of HSP27. The scale bars correspond to the non-expanded dimensions, derived by dividing by the sample expansion factor of 4.3. (D) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples (n = 30). The error bar is the standard deviation for the different cytoplasmic regions. (E) Distribution of the intensity of individual spots (n = 3342) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots.


( F ) Immunostaining of HSP27 ( white ) in motor neurons differentiated from CMT patient - derived iPSCs that harbor the heterozygous P182L mutation reveals large , cytoplasmic aggregates ( center and right panels ) . The control motor neurons that contain WT HSP27 do not show evidence of any aggregates . The nucleus is stained with Hoechst ( blue ) . Scale bar , 10 µm .

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(F) Immunostaining of HSP27 (white) in motor neurons differentiated from CMT patient-derived iPSCs that harbor the heterozygous P182L mutation reveals large, cytoplasmic aggregates (center and right panels). The control motor neurons that contain WT HSP27 do not show evidence of any aggregates. The nucleus is stained with Hoechst (blue). Scale bar, 10 µm.


( A ) Primary sequences of the WT peptide ( red ) and P182L peptide ( blue ) . The IxI / V motif is indicated with white text . Below : regions of 2D 1H - 15N HSQC spectra of 15N - labeled cHSP27 ( black ) in the presence of increasing amounts of WT ( red ) or P182L peptide ( blue ) . Resonances in the β4 and β8 strands broaden and disappear upon peptide binding , indicative of intermediate exchange . ( B ) Zoomed - in regions of resonances during the peptide titrations . The color bars indicate the amount of added WT or P182L peptide . ( C ) The combined 1HN and 15N chemical shift changes , denoted as chemical shift perturbation ( CSP ) , are plotted as a function of added peptide concentration . ( D ) CSPs shown as a function of residue number for the WT ( red ) and P182L peptide ( blue ) . ( E ) X - ray structure of cHSP27 bound to the WT peptide ( PDB : 4mjh ) , shown in white sticks , with the results from ( D ) plotted onto the structure .

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(A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.


( A , B ) i . 15N CPMG RD data for residues in the β4 strand ( T113 ) and the β8 strand ( S154 ) in the absence of added peptide . CPMG data are shown for the same residues in the presence of ii . ca . 2 % cHSP27 - WT peptide complex or iii . ca . 2 % of the cHSP27 - P182L peptide complex . Uncertainties in R2 , eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values . ( C ) i . The association ( kon ) and dissociation ( koff ) rates for WT ( red ) and P182L ( blue ) IxI / V peptide binding to the ACD , calculated directly from the CPMG RD data . Residues that are impacted by the presence of a small amount of peptide are indicated by red ( ii . , WT ) and blue ( iii . , P182L ) spheres . In both cases , residues in similar ACD regions show enhanced Rex , indicative of chemical exchange at similar sites .

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(A, B) i. 15N CPMG RD data for residues in the β4 strand (T113) and the β8 strand (S154) in the absence of added peptide. CPMG data are shown for the same residues in the presence of ii. ca. 2% cHSP27-WT peptide complex or iii. ca. 2% of the cHSP27-P182L peptide complex. Uncertainties in R2,eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values. (C) i. The association (kon) and dissociation (koff) rates for WT (red) and P182L (blue) IxI/V peptide binding to the ACD, calculated directly from the CPMG RD data. Residues that are impacted by the presence of a small amount of peptide are indicated by red (ii., WT) and blue (iii., P182L) spheres. In both cases, residues in similar ACD regions show enhanced Rex, indicative of chemical exchange at similar sites.


( B ) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control , obtained with affinity - enrichment mass spectrometry . Proteins that co - immunoprecipitated significantly more ( p < 0 . 05 ) with the P182L variant are shown in the upper right quadrant . Interactors with one or more [ I / V ] - x - [ I / V ] motifs are displayed in red while other significantly enriched interactors are displayed in black .

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(B) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control, obtained with affinity-enrichment mass spectrometry. Proteins that co-immunoprecipitated significantly more (p<0.05) with the P182L variant are shown in the upper right quadrant. Interactors with one or more [I/V]-x-[I/V] motifs are displayed in red while other significantly enriched interactors are displayed in black.


( C - D ) Co - immunoprecipitation from HeLa cells stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) using anti - V5 beads . Co - immunoprecipitation was quantified and presented as a percentage relative to the P182L variant ( n = 3 ) . The mean ± one standard deviation ( n = 3 ) is shown . Right : the location of Pro182 ( red ) in the crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) and in silico creation of the P182L mutation ( blue ) . The peptide is shown in white sticks . P182L specifically weakens binding of the HSP27 IxI / V motif .

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(C-D) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) using anti-V5 beads. Co-immunoprecipitation was quantified and presented as a percentage relative to the P182L variant (n=3). The mean ± one standard deviation (n=3) is shown. Right: the location of Pro182 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the P182L mutation (blue). The peptide is shown in white sticks. P182L specifically weakens binding of the HSP27 IxI/V motif.


( E ) Co - immunoprecipitation from HeLa cells stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) and transiently transfected with BAG3 - eGFP ( WT or IPV - mutant ) using anti - GFP beads . Western blots for anti - GFP ( BAG3 ) , V5 ( HSPB1 ) , and α - tubulin . NS indicates a non - specific band . Note that detection of BAG3 ( anti - GFP ) and HSP27 ( anti - V5 ) occurs with different antibodies . The locations of the IPV - to - GPG mutations in BAG3 are depicted below .

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(E) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) and transiently transfected with BAG3-eGFP (WT or IPV-mutant) using anti-GFP beads. Western blots for anti-GFP (BAG3), V5 (HSPB1), and α-tubulin. NS indicates a non-specific band. Note that detection of BAG3 (anti-GFP) and HSP27 (anti-V5) occurs with different antibodies. The locations of the IPV-to-GPG mutations in BAG3 are depicted below.


( F ) Co - immunoprecipitation from HeLa cells transiently transfected with V5 - epitope - tagged HSP27 ( P182L or S155Q / P182L mutant ) using anti - V5 beads ( n = 2 ) . Right : the location of Ser155 ( red ) in the crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) and in silico creation of the S155Q mutation ( purple ) showing a clash with the bound peptide . The peptide is shown in white sticks . S155Q disrupts binding of all IxI / V motifs .

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(F) Co-immunoprecipitation from HeLa cells transiently transfected with V5-epitope-tagged HSP27 (P182L or S155Q/P182L mutant) using anti-V5 beads (n=2). Right: the location of Ser155 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the S155Q mutation (purple) showing a clash with the bound peptide. The peptide is shown in white sticks.S155Q disrupts binding of all IxI/V motifs.


( D ) Immunostaining of PGCLCs d5 cryosections for SOX2 ( magenta ) and TFAP2C ( cyan ) . Barplot indicates the quantification of SOX2 + cells , TFAP2C + cells , and SOX2 + / TFAP2C + . n = 1150 cells , from n = 3 separate inductions , using two biological clones . The white squares represent the position of the magnified region at the bottom . Scale bar , 50 µm and 10 µm for the magnified region .

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(D) Immunostaining of PGCLCs d5 cryosections for SOX2 (magenta) and TFAP2C (cyan). Barplot indicates the quantification of SOX2+ cells, TFAP2C+ cells, and SOX2+/TFAP2C+. n=1150 cells, from n=3 separate inductions, using two biological clones. The white squares represent the position of the magnified region at the bottom. Scale bar, 50 µm and 10 µm for the magnified region.


( C ) MA plot of differential gene expression changes between XGFP - and XGFP + PGCLCs as determined by RNA - seq . Log2 - mean expression ( log2 - normalised counts from DESeq2 ) on the x - axis and the log2 - fold change on the y - axis are shown . Significantly upregulated and downregulated genes are highlighted in red and green respectively . False Discovery Rate ( FDR ) < 0 . 001 . Non - significant genes with log2 - mean expression between 0 and 0 . 2 were removed for easier plot visualisation .

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(C) MA plot of differential gene expression changes between XGFP- and XGFP+ PGCLCs as determined by RNA-seq. Log2-mean expression (log2-normalised counts from DESeq2) on the x-axis and the log2-fold change on the y-axis are shown. Significantly upregulated and downregulated genes are highlighted in red and green respectively. False Discovery Rate (FDR) < 0.001. Non-significant genes with log2-mean expression between 0 and 0.2 were removed for easier plot visualisation.


( D ) Expression levels ( normalised DEseq2 counts ) of selected differentially expressed genes between XGFP - and XGFP + PGCLCs during the differentiation time course . Genes with FDR < 0 . 001 were considered significantly differentially expressed . Points indicate expression of individual biological replicates .

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(D) Expression levels (normalised DEseq2 counts) of selected differentially expressed genes between XGFP- and XGFP+ PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.


( E ) Heatmap of RNA - seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side .

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(E) Heatmap of RNA-seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side.


( G ) FACS analysis of cell cycle using DAPI . Numbers indicate the percentage of cells in G1 , S and G2 / M respectively .

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(G) FACS analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.


( H ) Alkaline phosphatase staining for ESCs , XGFP + PGCLCs and XGFP - PGCLCs grown for 7 days in 2i / LIF medium on immortalised mouse embryonic fibroblasts . ( I ) Barplot indicating the absolute numbers of Alkaline Phosphatase ( AP ) positive colonies in each cell type after 7 days of culture in 2i / LIF medium on immortalised mouse embryonic fibroblasts . Y - axis is in square root scale ( sqrt ) for better plot visualisation . Each white dot represents one technical replicate .

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(H) Alkaline phosphatase staining for ESCs, XGFP+ PGCLCs and XGFP- PGCLCs grown for 7 days in 2i/LIF medium on immortalised mouse embryonic fibroblasts. (I) Barplot indicating the absolute numbers of Alkaline Phosphatase (AP) positive colonies in each cell type after 7 days of culture in 2i/LIF medium on immortalised mouse embryonic fibroblasts. Y-axis is in square root scale (sqrt) for better plot visualisation. Each white dot represents one technical replicate.


( H ) Immunofluorescence images of SYCP3 ( red ) , DAZL ( yellow ) and DAPI at agg11 of IVDi tissue maturation from XGFP - PGCLCs . IVDi = in vitro differentiation . White squares indicate the positions of the magnified section shown below . Top panel scale bar = 100 μm . Middle panel scale bar = 10 μm . Bottom panel scale bar = 50 μm . ( I ) Quantification of SYCP3 + cells ( oocytes in cyst and primary follicles ) in IVDi tissues at agg11 . Each dot represents one IVDi tissue performed in 3 biological replicates .

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(H) Immunofluorescence images of SYCP3 (red), DAZL (yellow) and DAPI at agg11 of IVDi tissue maturation from XGFP- PGCLCs. IVDi = in vitro differentiation. White squares indicate the positions of the magnified section shown below. Top panel scale bar = 100 μm. Middle panel scale bar = 10 μm. Bottom panel scale bar = 50 μm. (I) Quantification of SYCP3+ cells (oocytes in cyst and primary follicles) in IVDi tissues at agg11. Each dot represents one IVDi tissue performed in 3 biological replicates.


( A ) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP - and XGFP + PGCLCs . Meiosis is induced via addition of Retinoic Acid ( RA ) and Bone Morphogenetic Protein 2 ( BMP2 ) . c0 = starting day of culture , c9 = culture day 9 and last day of culture . ( B ) Representative images for the expression of XGFP ( green ) and SYCP3 ( red ) in germ cells at c5 , c7 and c9 from XGFP + and XGFP - PGCLCs . All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3 + cells . Cells were counterstained with DAPI ( grey ) . Scale bars = 10 μm . ( C ) Number of SYCP3 + cells per m220 culture day originating from XGFP + and XGFP - PGCLCs . Each white dot represents a biological replicate ( n = 3 ) . P - values shown are from a two - sided Mann - Whitney U test . n . s . = not significant . ( D ) Percentage of XGFP + cells among SYCP3 + cells at the indicated m220 culture day , originating from XGFP + or XGFP - PGCLCs . Green bars show constitutive GFP expression in XGFP + PGCLC - derived cells , while striped bars signify XGFP reactivation in XGFP - cells . Each white dot represents a biological replicate ( n = 3 ) .

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(A) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP- and XGFP+ PGCLCs. Meiosis is induced via addition of Retinoic Acid (RA) and Bone Morphogenetic Protein 2 (BMP2). c0 = starting day of culture, c9 = culture day 9 and last day of culture. (B) Representative images for the expression of XGFP (green) and SYCP3 (red) in germ cells at c5, c7 and c9 from XGFP+ and XGFP- PGCLCs. All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3+ cells. Cells were counterstained with DAPI (grey). Scale bars = 10 μm. (C) Number of SYCP3+ cells per m220 culture day originating from XGFP+ and XGFP- PGCLCs. Each white dot represents a biological replicate (n = 3). P-values shown are from a two-sided Mann-Whitney U test. n.s. = not significant. (D) Percentage of XGFP+ cells among SYCP3+ cells at the indicated m220 culture day, originating from XGFP+ or XGFP- PGCLCs. Green bars show constitutive GFP expression in XGFP+ PGCLC-derived cells, while striped bars signify XGFP reactivation in XGFP- cells. Each white dot represents a biological replicate (n = 3).


( E ) Representative images showing stages of meiotic prophase I from culture day 9 ( c9 ) germ cells from XGFP + and XGFP - PGCLCs . c9 germ cells were spread and immunostained for SYCP3 ( red ) , and γH2AX ( grey ) . Scale bars = 10 µm . ( F ) Quantification of meiotic progression in culture day ( c9 ) expanded germ cells derived from XGFP + and XGFP - PGCLCs . The graphs show the percentages of the meiotic stage for SYCP3 + cells . Lepto , leptotene ; Zygo , zygotene ; Pachy , pachytene ; Diplo , diplotene . Numbers indicate detected cells per meiotic stage and original XGFP status .

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(E) Representative images showing stages of meiotic prophase I from culture day 9 (c9) germ cells from XGFP+ and XGFP- PGCLCs. c9 germ cells were spread and immunostained for SYCP3 (red), and γH2AX (grey). Scale bars = 10 µm. (F) Quantification of meiotic progression in culture day (c9) expanded germ cells derived from XGFP+ and XGFP- PGCLCs. The graphs show the percentages of the meiotic stage for SYCP3+ cells. Lepto, leptotene; Zygo, zygotene; Pachy, pachytene; Diplo, diplotene. Numbers indicate detected cells per meiotic stage and original XGFP status.


( G ) Representative tile scan merged images of XGFP ( green ) and Xtomato ( red ) in germ cells at c7 from XGFP + and XGFP - PGCLCs . Cells were counterstained with DAPI ( grey ) . Scale Bar = 1 mm . The white squares contain a magnified image of the region depicted by the white dotted squares ( Scale Bar = 1 µm ) . ( H ) Density ridge plot showing size of cellular aggregates in ( G ) measured using the XTomato signal . X - Axis shows area in µm2 scaled as log2 . Dashed line represents the mean . XFGP - mean = 1320 µm2 , XGFP + mean = 5514 µm2 . XGFP - n = 297 aggregates , XGFP + n = 373 aggregates . P - value from two - sample unpaired Wilcoxon - Mann - Whitney test with R defaults .

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(G) Representative tile scan merged images of XGFP (green) and Xtomato (red) in germ cells at c7 from XGFP+ and XGFP- PGCLCs. Cells were counterstained with DAPI (grey). Scale Bar = 1 mm. The white squares contain a magnified image of the region depicted by the white dotted squares (Scale Bar = 1 µm). (H) Density ridge plot showing size of cellular aggregates in (G) measured using the XTomato signal. X-Axis shows area in µm2 scaled as log2. Dashed line represents the mean. XFGP- mean = 1320 µm2, XGFP+ mean = 5514 µm2. XGFP- n = 297 aggregates, XGFP+ n = 373 aggregates. P-value from two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults.


( A ) Cellomics images ( top ) of GFP - LC3 ‐ HEK cells in full medium ( FM ) or after 2 h in starvation medium ( ES ) with leupeptin ( EL ) . Scale bars = 40 μm . Optimised automated analysis ( bottom left ) programme detecting hoechst ‐ labelled nuclei ( purple line ) , cell outline ( red line ) and identifies GFP - LC3 spots ( red spots ) in FM and EL . Enlarged GFP - LC3 images in FM and EL ( bottom right ) . Scale bars = 10 μm .

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(A) Cellomics images (top) of GFP-LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP-LC3 spots (red spots) in FM and EL. Enlarged GFP-LC3 images in FM and EL (bottom right). Scale bars=10 μm.


( B ) SCPO , STIPO and STAPO after indicated siRNA treatment of GFP - LC3 ‐ HEK cells incubated as in ( A ) . Error bars represent s . e . m . Significance was determined using a two ‐ tailed paired t ‐ test compared with RISCfree ( RF ) in EL : SCPO siULK1 , * * * P = 0 . 0002 ; siATG7 , * * P = 0 . 0078 ; siNRBP2 , * * P = 0 . 0088 . STIPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0060 ; siNRBP2 , * * P = 0 . 0041 . STAPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0037 ; siNRBP2 , * * P = 0 . 0028 . The experiments were performed : RISCfree ( n = 5 ) , siULK1 ( n = 5 ) , siATG7 ( n = 3 ) , siNRBP2 ( n = 3 ) .

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(B) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP-LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3).


( D ) Scatter plot of median SCPO Z ‐ scores from siRNA pools in the primary screen that met the minimum object count ( OC ) cutoff . Dashed lines represent approximate cutoff for 500 best increasers and decreasers . RP of known autophagy genes ( ATG4C , ATG14 , RB1CC1 , ATG9A , ATG7 ) are given in Supplementary Table S2 ; AMBRA1 ( FLJ20294 ) , TBC1D25 ( OATL1 ) and SH3GLB1 ( BIF ‐ 1 ) in Supplementary Table S1 ; LAMTOR2 has a RP = 708 . 7 and RUBICON has a RP = 1126 .

(D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126.


( A ) SCPO , STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP - LC3 spot phenotype , above a cutoff of greater or less than 20 % of control , and passing additional criteria ( Supplementary Table 3 ) . Red boxes indicate an increase > 1 . 2 ; pink boxes an increase between 1 . 1 and 1 . 2 ; white boxes are between 0 . 9 and 1 . 1 ; light blue boxes a decrease between 0 . 9 and 0 . 8 ; dark blue boxes a decrease to 0 . 8 . Hits underlined were taken forward as three out of four4 hits , ( § ) not chosen because of restricted tissue expression , ( * ) are three out of four when alternate method of normalisation is used .

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(A) SCPO, STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP-LC3 spot phenotype, above a cutoff of greater or less than 20% of control, and passing additional criteria (Supplementary Table 3). Red boxes indicate an increase >1.2; pink boxes an increase between 1.1 and 1.2; white boxes are between 0.9 and 1.1; light blue boxes a decrease between 0.9 and 0.8; dark blue boxes a decrease to 0.8. Hits underlined were taken forward as three out of four4 hits, (§) not chosen because of restricted tissue expression, (*) are three out of four when alternate method of normalisation is used.


( B ) Table summarising results from further validation of 20 genes . mRNA levels scored as ( + ) or ( − ) indicate efficient or poor knockdown , respectively ( see Supplementary Figures S1 and S2 ) . Increasers ( left ) or decreasers ( right ) assayed for LC3 lipidation were scored ( + ) for yes , ( − ) for no , ( + / − ) subtle change and ( nd ) not done ( Supplementary Figure S3 ) .

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(B) Table summarising results from further validation of 20 genes. mRNA levels scored as (+) or (−) indicate efficient or poor knockdown, respectively (see Supplementary Figures S1 and S2). Increasers (left) or decreasers (right) assayed for LC3 lipidation were scored (+) for yes, (−) for no, (+/−) subtle change and (nd) not done (Supplementary Figure S3).


( C - E ) STIPO results after incubation under different autophagy ‐ inducing conditions following ( C ) SCOC knockdown ( D ) SUPT5H knockdown and ( E ) WAC knockdown . siRNA ‐ treated cells were incubated for the indicated time in FL : full medium plus leupeptin ; ES ; EL : ES plus leupeptin ; Torin : 250 nM Torin1 in FL ; Resv : 128 μM Resveratrol in FL ; LiCl : 10 mM LiCl in FM .

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(C-E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM.


( B ) Endogenous SCOC shows colocalisation with Atg9 ( red ) and TGN46 ( blue ) . HEK293 cells were fixed and permeabilised in saponin . Colocalisation indicated with arrows .

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(B) Endogenous SCOC shows colocalisation with Atg9 (red) and TGN46 (blue). HEK293 cells were fixed and permeabilised in saponin. Colocalisation indicated with arrows.


( C ) Endogenous SCOC ( green top , red bottom ) partially colocalises with endogenous LC3 ( red , top ) and GFP - LC3 ( green , bottom ) in HEK293 and GFP - LC3 ‐ HEK cells , respectively , after 2 h amino ‐ acid starvation . Boxed region is at higher magnification in merge panel on right . Scale bars = 10 μm .

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(C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP-LC3 (green, bottom) in HEK293 and GFP-LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm.


( A ) SCOC is required for autophagy in HEK293 cells . Anti ‐ ULK1 , ‐ Actin and ‐ LC3 blot after siRNA treatment in HEK293 cells in FM , ES or EL . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RF EL versus siSCOC ‐ 03 EL , * P = 0 . 0419 .

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(A) SCOC is required for autophagy in HEK293 cells. Anti‐ULK1, ‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siSCOC‐03 EL, *P=0.0419.


( B ) SCOC is required for p62 degradation . p62 levels were determined by western blot ( Supplementary Figure S5 ) after incubation in FM , ES for 2 or 4 h , or ES with BafilomycinA1 ( ES + BAF ) for 2 and 4 h . Quantification of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree ES 2 and 4 h , * * * P > 0 . 0001 and * * 0 . 0014 , respectively .

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(B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively.


( C ) SCOC - GFP and SCOC - myc levels are reduced by SCOC siRNA duplex ‐ 03 . Anti ‐ GFP , ‐ Myc and ‐ Actin blots of HEK293 cells after treatment with ULK1 , RISCfree or SCOC siRNA and overexpression of GFP , SCOC - myc or SCOC - GFP .

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(C) SCOC-GFP and SCOC-myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC-myc or SCOC-GFP.


( D ) Overexpression of SCOC - myc increases autophagy . Anti ‐ Actin , ‐ LC3 and ‐ Myc blots of HEK293 cells after transfection of pcDNA or SCOC - myc . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 5 ) . SCOC - myc EL versus pcDNA EL , * P = 0 . 0215 .

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(D) Overexpression of SCOC-myc increases autophagy. Anti‐Actin, ‐LC3 and ‐Myc blots of HEK293 cells after transfection of pcDNA or SCOC-myc. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=5). SCOC-myc EL versus pcDNA EL, *P=0.0215.


( E ) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells . Arrows show partial colocalisation . Boxed region is at higher magnification in merge panel on bottom right . Scale bars = 20 μm .

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(E) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells. Arrows show partial colocalisation. Boxed region is at higher magnification in merge panel on bottom right. Scale bars=20 μm.


( A ) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation . Anti ‐ Actin and ‐ LC3 blot after siRNA treatment in HEK293 cells . Quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RISCfree ES versus siFEZ1 ‐ 01 ES , P = 0 . 034 . Quantification of p62 / actin ( Supplementary Figure S5 ) performed as in Figure 4B ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree 2 and 4 hP > 0 . 0001 and P = 0 . 0014 , respectively . qRT - PCR showing FEZ1 mRNA levels after indicated knockdown in GFP - LC3 ‐ HEK cells ( error bars represent s . d . ) .

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(A) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation. Anti‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells. Quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RISCfree ES versus siFEZ1‐01 ES, P=0.034. Quantification of p62/actin ( Supplementary Figure S5) performed as in Figure 4B; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree 2 and 4 hP>0.0001 and P=0.0014, respectively. qRT-PCR showing FEZ1 mRNA levels after indicated knockdown in GFP-LC3‐HEK cells (error bars represent s.d.).


( B ) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation . Anti ‐ Myc , ‐ Actin and ‐ LC3 blot after transfection with pcDNA or Myc - FEZ1 in HEK293 cells . Quantification of LC3II / actin ; error bars represent s . e . m . ( n = 3 ) ; pcDNA ES versus Myc - FEZ1 ES , P = 0 . 0318 . Anti ‐ GFP , ‐ Actin and ‐ p62 blot after transfection with GFP or FEZ1 - GFP and treatment with ES or ES plus Bafilomycin ( EB ) for 4 h . Quantification of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; GFP FM versus GFP ES , P = 0 . 0077 ; GFP FM versus GFP EB , P = 0 . 0212 ; GFP ES versus FEZ1 - GFP ES , P = 0 . 048 .

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(B) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation. Anti‐Myc, ‐Actin and ‐LC3 blot after transfection with pcDNA or Myc-FEZ1 in HEK293 cells. Quantification of LC3II/actin; error bars represent s.e.m. (n=3); pcDNA ES versus Myc-FEZ1 ES, P=0.0318. Anti‐GFP, ‐Actin and ‐p62 blot after transfection with GFP or FEZ1-GFP and treatment with ES or ES plus Bafilomycin (EB) for 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); GFP FM versus GFP ES, P=0.0077; GFP FM versus GFP EB, P=0.0212; GFP ES versus FEZ1-GFP ES, P=0.048.


( C ) SCOC and FEZ1 interact . Left panel shows anti ‐ SCOC and ‐ FEZ1 blots after co ‐ expression in HEK293 cells and co ‐ immunoprecipitation of SCOC - myc and FEZ1 - GFP with anti ‐ GFP antibody . Input lysate ( Inp ) , unbound supernatant ( Unb ) . Right panel shows anti ‐ FEZ1 and ‐ FLAG blots after co ‐ expression in HEK293 cells and co ‐ immunoprecipitation of FLAG - SCOC and Myc - FEZ1 with anti ‐ FLAG antibody .

(C) SCOC and FEZ1 interact. Left panel shows anti‐SCOC and ‐FEZ1 blots after co‐expression in HEK293 cells and co‐immunoprecipitation of SCOC-myc and FEZ1-GFP with anti‐GFP antibody. Input lysate (Inp), unbound supernatant (Unb). Right panel shows anti‐FEZ1 and ‐FLAG blots after co‐expression in HEK293 cells and co‐immunoprecipitation of FLAG-SCOC and Myc-FEZ1 with anti‐FLAG antibody.


( D ) FEZ1 interacts with SCOC in vitro . GST ‐ pulldown assays using in vitro translated 35S ‐ labelled Myc - FEZ1 ( right ) or 35S ‐ labelled Myc ‐ control ( left ) and GST or GST - SCOC ( asterisk in bottom panel ) . Bound proteins were detected by autoradiography following SDS - PAGE ( top ) , protein loading by coomassie blue ( bottom ) .

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(D) FEZ1 interacts with SCOC in vitro. GST‐pulldown assays using in vitro translated 35S‐labelled Myc-FEZ1 (right) or 35S‐labelled Myc‐control (left) and GST or GST-SCOC (asterisk in bottom panel). Bound proteins were detected by autoradiography following SDS-PAGE (top), protein loading by coomassie blue (bottom).


( E ) The FEZ1 - SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled ‐ coil domain of FEZ1 as demonstrated in GST ‐ pulldown assays with in vitro translated 35S ‐ labelled WT or mutant Myc ‐ FEZ1 and GST or GST ‐ SCOC . Autoradiographs of 10 % input and bound proteins ( top panels ) and Coomassie blue ‐ stained gels of bead ‐ bound GST and GST ‐ SCOC proteins used ( bottom panels ) .

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(E) The FEZ1-SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled‐coil domain of FEZ1 as demonstrated in GST‐pulldown assays with in vitro translated 35S‐labelled WT or mutant Myc‐FEZ1 and GST or GST‐SCOC. Autoradiographs of 10% input and bound proteins (top panels) and Coomassie blue‐stained gels of bead‐bound GST and GST‐SCOC proteins used (bottom panels).


( A ) FEZ1 interacts with both the kinase domain ( 1 - 278 ) and the proline - serine rich spacer of ULK1 ( 279 - 828 ) . GFP ‐ or Myc ‐ tagged ULK1 fragments were in vitro translated with 35S ‐ methionine and subjected to GST ‐ pulldown assays using GST and GST - FEZ1 purified from E . coli ( bottom ) . Bound proteins were detected by autoradiography following SDS - PAGE ( top ) .

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(A) FEZ1 interacts with both the kinase domain (1-278) and the proline-serine rich spacer of ULK1 (279-828). GFP‐ or Myc‐tagged ULK1 fragments were in vitro translated with 35S‐methionine and subjected to GST‐pulldown assays using GST and GST-FEZ1 purified from E. coli (bottom). Bound proteins were detected by autoradiography following SDS-PAGE (top).


( B ) ULK1 interacts with FEZ1 . ULK1 was immunoprecipitated from HEK293 lysates with anti ‐ ULK1 and bound ULK1 and FEZ1 were detected with anti ‐ ULK1 and anti ‐ FEZ1 antibodies . Input lysate ( Inp ) .

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(B) ULK1 interacts with FEZ1. ULK1 was immunoprecipitated from HEK293 lysates with anti‐ULK1 and bound ULK1 and FEZ1 were detected with anti‐ULK1 and anti‐FEZ1 antibodies. Input lysate (Inp).


( C ) The FEZ1 - GFP and Myc - ULK1 interaction is unaffected by amino ‐ acid starvation . Anti ‐ Myc and ‐ GFP blots after HEK293 cells were co ‐ transfected with FEZ1 - GFP and Myc - ULK1 , incubated in FM or ES for 2 h , harvested and the complex was immunoprecipitated using anti ‐ GFP antibody .

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(C) The FEZ1-GFP and Myc-ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1-GFP and Myc-ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody.


( D ) BN - PAGE reveals SCOC and FEZ1 are in a complex . FEZ1 - GFP , FLAG - SCOC and Myc - ULK1 wild type or kinase ‐ inactive ULK1K46I ( ULK1KI ) were co ‐ expressed in HEK293 cells . Anti ‐ GFP , ‐ FLAG and ‐ Myc western blots are shown for the BN - PAGE gel ( top ) or the SDS - PAGE gel ( bottom ) . In GFP and FLAG western blots , two different exposures of the same BN - PAGE blot are shown spliced together .

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(D) BN-PAGE reveals SCOC and FEZ1 are in a complex. FEZ1-GFP, FLAG-SCOC and Myc-ULK1 wild type or kinase‐inactive ULK1K46I (ULK1KI) were co‐expressed in HEK293 cells. Anti‐GFP, ‐FLAG and ‐Myc western blots are shown for the BN-PAGE gel (top) or the SDS-PAGE gel (bottom). In GFP and FLAG western blots, two different exposures of the same BN-PAGE blot are shown spliced together.


( E ) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC . RF or ULK1 siRNA ‐ treated cells were co ‐ transfected with FEZ1 - GFP and FLAG - SCOC , then incubated and immunoprecipitated as in ( C ) . Western blot of lysates for ULK1 , GFP and FLAG , and immunoprecipitates with GFP and FLAG antibodies .

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(E) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC. RF or ULK1 siRNA‐treated cells were co‐transfected with FEZ1-GFP and FLAG-SCOC, then incubated and immunoprecipitated as in (C). Western blot of lysates for ULK1, GFP and FLAG, and immunoprecipitates with GFP and FLAG antibodies.


( F ) siRNA depletion of SCOC reduces FEZ1 - ULK1 complex . HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1 - GFP . FEZ1 - GFP was detected with anti ‐ GFP in both BN - PAGE and SDS - PAGE . Endogenous ULK1 was detected with anti ‐ ULK1 antibody . Arrowheads indicate a FEZ1 - GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex > 1236 kDa also detected in ( D ) in the triple transfection . The 200 KDa complex is sensitive to loss of SCOC and ULK1 .

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(F) siRNA depletion of SCOC reduces FEZ1-ULK1 complex. HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1-GFP. FEZ1-GFP was detected with anti‐GFP in both BN-PAGE and SDS-PAGE. Endogenous ULK1 was detected with anti‐ULK1 antibody. Arrowheads indicate a FEZ1-GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex >1236 kDa also detected in (D) in the triple transfection. The 200 KDa complex is sensitive to loss of SCOC and ULK1.


( G ) SCOC can reduce the interaction of ULK1 with MBP - FEZ1 . Immobilised MBP - FEZ1 was incubated with in vitro 35S ‐ methionine ‐ labelled Myc - ULK1 . Addition of increasing amounts of in vitro translated 35S ‐ methionine SCOC reduced the amount of Myc - ULK1 bound . Top and bottom panels are as described in ( A ) .

(G) SCOC can reduce the interaction of ULK1 with MBP-FEZ1. Immobilised MBP-FEZ1 was incubated with in vitro 35S‐methionine‐labelled Myc-ULK1. Addition of increasing amounts of in vitro translated 35S‐methionine SCOC reduced the amount of Myc-ULK1 bound. Top and bottom panels are as described in (A).


( A ) SCOC interacts with UVRAG . HEK293 cells were transfected with FLAG - SCOC and Myc - UVRAG , lysed and incubated with anti ‐ FLAG ( left ) or anti ‐ Myc ( right ) antibodies . Co ‐ immunoprecipitation of Myc - UVRAG ( left ) and FLAG - SCOC ( right ) was confirmed using anti ‐ Myc and anti ‐ FLAG antibodies for western blotting . Asterisk in ( A - C ) and ( D ) indicates non ‐ specific bands .

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(A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG-SCOC and Myc-UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc-UVRAG (left) and FLAG-SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A-C) and (D) indicates non‐specific bands.


( B ) Interaction of SCOC with UVRAG is unaffected by FEZ1 - GFP . As in ( A ) , co ‐ immunoprecipitation was preformed in HEK293 cells transfected with FLAG - SCOC , Myc - UVRAG and FEZ1 - GFP . Myc - UVRAG was detected as in ( A ) , FEZ1 - GFP was detected with anti ‐ GFP antibody .

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(B) Interaction of SCOC with UVRAG is unaffected by FEZ1-GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG-SCOC, Myc-UVRAG and FEZ1-GFP. Myc-UVRAG was detected as in (A), FEZ1-GFP was detected with anti‐GFP antibody.


( C ) The SCOC and UVRAG interaction is reduced by starvation , and this reduction is inhibited by FEZ1 - GFP . HEK293 cells were transfected as in ( B ) and 24 h later either treated with FM or ES for 2 h . FLAG - SCOC or Myc - UVRAG was immunoprecipitated with anti ‐ FLAG or anti ‐ Myc antibodies and the bound proteins were detected with anti ‐ Myc , ‐ GFP or ‐ FLAG antibodies by western blotting . Quantification of the amount of FLAG - SCOC co ‐ immunoprecipitated with Myc - UVRAG in FM or ES , without overexpressed FEZ1 - GFP ( left ) or with overexpressed FEZ1 - GFP ( right ) . Significance was determined using a two ‐ tailed paired t ‐ test ( n = 3 ) .

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(C) The SCOC and UVRAG interaction is reduced by starvation, and this reduction is inhibited by FEZ1-GFP. HEK293 cells were transfected as in (B) and 24 h later either treated with FM or ES for 2 h. FLAG-SCOC or Myc-UVRAG was immunoprecipitated with anti‐FLAG or anti‐Myc antibodies and the bound proteins were detected with anti‐Myc, ‐GFP or ‐FLAG antibodies by western blotting. Quantification of the amount of FLAG-SCOC co‐immunoprecipitated with Myc-UVRAG in FM or ES, without overexpressed FEZ1-GFP (left) or with overexpressed FEZ1-GFP (right). Significance was determined using a two‐tailed paired t‐test (n=3).


( D ) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction . HEK293 cells were transfected with FEZ1 - GFP , GFP or FLAG - SCOC alone or in combination . Top panel input , bottom panel immunoprecipitation with anti ‐ GFP or anti ‐ FLAG . Western blot for endogenous UVRAG , GFP - FEZ1 GFP or FLAG - SCOC as indicated . Arrowhead : endogenous UVRAG .

(D) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction. HEK293 cells were transfected with FEZ1-GFP, GFP or FLAG-SCOC alone or in combination. Top panel input, bottom panel immunoprecipitation with anti‐GFP or anti‐FLAG. Western blot for endogenous UVRAG, GFP-FEZ1 GFP or FLAG-SCOC as indicated. Arrowhead: endogenous UVRAG.


( E ) UVRAG can interact with both FEZ1 and SCOC in vitro . GST , GST - FEZ1 or GST - SCOC were immobilised and incubated with 35S ‐ labelled Myc - UVRAG as indicated . Arrowheads indicate GST - FEZ1 ( left ) and GST - SCOC ( right ) .

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(E) UVRAG can interact with both FEZ1 and SCOC in vitro. GST, GST-FEZ1 or GST-SCOC were immobilised and incubated with 35S‐labelled Myc-UVRAG as indicated. Arrowheads indicate GST-FEZ1 (left) and GST-SCOC (right).


( F ) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells . Arrows show colocalisation . Scale bar = 10 μm .

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(F) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells. Arrows show colocalisation. Scale bar=10 μm.


( A ) Depletion of endogenous WAC . Anti ‐ WAC and ‐ Actin blot and WAC mRNA levels by qRT - PCR after treatment with RF or individual WAC siRNAs ( W ‐ 01 to W ‐ 04 ) in HEK293 cells . qRT - PCR error bars represent standard deviation of triplicates ( n = 2 ) .

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(A) Depletion of endogenous WAC. Anti‐WAC and ‐Actin blot and WAC mRNA levels by qRT-PCR after treatment with RF or individual WAC siRNAs (W‐01 to W‐04) in HEK293 cells. qRT-PCR error bars represent standard deviation of triplicates (n=2).


( B ) ( B ) WAC is required for autophagy . Anti ‐ ULK1 , ‐ WAC , ‐ Actin and ‐ LC3 blots after siRNA treatment in HEK293 cells and incubation in FM , ES or EL . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RF EL versus siWAC ‐ 03 EL , * * P = 0 . 0079 .

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(B) (B) WAC is required for autophagy. Anti‐ULK1, ‐WAC, ‐Actin and ‐LC3 blots after siRNA treatment in HEK293 cells and incubation in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siWAC‐03 EL, **P=0.0079.


( C ) p62 degradation requires WAC . Anti ‐ p62 and ‐ Actin blot after siRNA treatment of GFP - LC3 ‐ HEK cells in FM or ES for 2 h in duplicate . Bars represent p62 / actin levels normalised to RF FM ; quantification of p62 / actin ; error bars represent s . e . m . ( n = 3 ) : RF FM versus RF ES , * * P = 0 . 0057 ; RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0025 ; RF ES versus siWAC ‐ 03 ES , * P = 0 . 0386 .

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(C) p62 degradation requires WAC. Anti‐p62 and ‐Actin blot after siRNA treatment of GFP-LC3‐HEK cells in FM or ES for 2 h in duplicate. Bars represent p62/actin levels normalised to RF FM; quantification of p62/actin; error bars represent s.e.m. (n=3): RF FM versus RF ES, **P=0.0057; RF FM versus siWAC‐03 FM, **P=0.0025; RF ES versus siWAC‐03 ES, *P=0.0386.


( D ) WAC depletion accelerates degradation of a UPS reporter . Anti ‐ p62 , ‐ GFP , ‐ Actin , ‐ LC3 , ‐ WAC and ‐ Atg16 blots after siRNA treatment in UbG76V - YFP MelJuSo cells and incubation conditions FM , ES or FM with 10 μM MG132 ( MG ) for 2 h . Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC ‐ 03 siRNA treatment ( n = 3 ) and compared with ATG16 siRNA treatment ( n = 2 ) . Error bars represent s . e . m . : RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0082 .

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(D) WAC depletion accelerates degradation of a UPS reporter. Anti‐p62, ‐GFP, ‐Actin, ‐LC3, ‐WAC and ‐Atg16 blots after siRNA treatment in UbG76V-YFP MelJuSo cells and incubation conditions FM, ES or FM with 10 μM MG132 (MG) for 2 h. Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC‐03 siRNA treatment (n=3) and compared with ATG16 siRNA treatment (n=2). Error bars represent s.e.m.: RF FM versus siWAC‐03 FM, **P=0.0082.