( B ) Plot of read pile - ups from MSM with inducible overexpression of MSMEG _ 0916 shows ChIP - binding in promoters of desA1 desA2 .

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(B) Plot of read pile-ups from MSM with inducible overexpression of MSMEG_0916 shows ChIP-binding in the promoters of desA1 and desA2.


Boxplots representing RPKM values from - seq of MSM with inducible overexpression of MSMEG _ 0916 . Significant log2 fold change ( FC ) between uninduced ( - ATc ) induced ( + ATc ) samples for MSMEG _ 0916 ( log2 FC = 4 . 99 ) , desA1 ( log2 FC = - 1 . 32 ) desA2 ( log2 FC = - 1 . 8 ) with multiple hypothesis adjusted P - values < 0 . 0001 . Data is from three biological samples ( induced ) nine biological samples ( uninduced ) .

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Boxplots representing RPKM values from RNA-seq of MSM with inducible overexpression of MSMEG_0916. Significant log2 fold change (FC) between uninduced (-ATc) and induced (+ATc) samples for MSMEG_0916 (log2 FC = 4.99), desA1 (log2 FC = -1.32) and desA2 (log2 FC = -1.8) with multiple hypothesis adjusted P-values < 0.0001. Data is from three biological samples (induced) and nine biological samples (uninduced).


( A ) Serial ten - fold dilutions of MTB H37Rv wild type ( wt ) MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc .

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(A) Serial ten-fold dilutions of MTB H37Rv wild type (wt) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc.


( B ) Argentation TLC of 14C - labeled methyl esters of mycolic ( MAMEs ) obtained from apolar lipids delipidated wall fractions of MSM wt MSM with inducible overexpression of MSMEG _ 0916 . α , αʹ , epoxy ( e ) cyclopropanated α - ( X1 ) MAMES species are labeled . Faster - migrating species that co - migrated with α - MAMES accumulate with induced MSMEG _ 0916 overexpression are indicated as X2 X3 .

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(B) Argentation TLC of 14C-labeled methyl esters of mycolic acids (MAMEs) obtained from apolar lipids and delipidated cell wall fractions of MSM wt and MSM with inducible overexpression of MSMEG_0916. The α, αʹ, epoxy (e) and cyclopropanated α- (X1) MAMES species are labeled. Faster-migrating species that co-migrated with α-MAMES and accumulate with induced MSMEG_0916 overexpression are indicated as X2 and X3.


( C ) BCG wt BCG with inducible overexpression of Rv0472c cultures , labeled with 14C - acetate , were induced ( + ATc ) or uninduced ( - ATc ) for 4 h or 8 h . total FAMEs MAMEs were extracted analyzed by autoradiography - TLC using equal ( 15 , 000 cpm ) for each lane .

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(C) BCG wt and BCG with inducible overexpression of Rv0472c cultures, labeled with 14C-acetate, were induced (+ATc) or uninduced (-ATc) for 4 h or 8 h. The total FAMEs and MAMEs were extracted and analyzed by autoradiography-TLC using equal counts (15,000 cpm) for each lane.


A , Volcano plot of differentially modulated in 22rv1 ( CRPC ) versus CWR22res ( HN ) tumours . Red blue dots represent that are significantly up - down - regulated , respectively ( p - value < 0 . 05 , FC = 1 . 5 ) . Data information : Data reproducibility : n = 3 tumours per group .

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A, Volcano plot of the differentially modulated proteins in 22rv1 (CRPC) versus CWR22res (HN) tumours. Red and blue dots represent the proteins that are significantly up- and down-regulated, respectively (p-value < 0.05, FC = 1.5). Data information: Data reproducibility: n = 3 tumours per group.


B , Western blot of THEM6 expression in CRPC ( 22rv1 LNCaP AI ) HN ( CWR22res LNCaP ) prostate orthografts . VCL was used as a sample loading control . Data information : Data reproducibility : : n = 1 gel loaded with three prostate orthografts per condition .

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B, Western blot analysis of THEM6 expression in CRPC (22rv1 and LNCaP AI) and HN (CWR22res and LNCaP) prostate orthografts. VCL was used as a sample loading control. Data information: Data reproducibility: : n = 1 gel loaded with three prostate orthografts per condition.


G , Tumour volume ( measured by ultrasound ) of CTL THEM6 KO 22rv1 - derived orthografts developed in surgically castrated mice . H , Tumour volume of CTL THEM6 KO LNCaP AI - derived xenografts developed in surgically castrated mice . I , Tumour volume ( measured by ultrasound ) of CTL THEM6 KO CWR22res - derived orthografts . Orchidectomy was performed 3 - after implantation . Data information : Data are presented as mean values + / - SD . Statistical : G , H : two - Mann - Whitney U . I : Kruskal - Wallis . Data reproducibility : G , I : n = 5 mice per group . Panel H : n = 7 mice per group .

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G, Tumour volume (measured by ultrasound) of CTL and THEM6 KO 22rv1-derived orthografts developed in surgically castrated mice. H, Tumour volume of CTL and THEM6 KO LNCaP AI-derived xenografts developed in surgically castrated mice. I, Tumour volume (measured by ultrasound) of CTL and THEM6 KO CWR22res-derived orthografts. Orchidectomy was performed 3-weeks after cell implantation. Data information: Data are presented as mean values +/- SD. Statistical analysis: G, H: two-tailed Mann-Whitney U test. I: Kruskal-Wallis test. Data reproducibility: G, I: n = 5 mice per group. Panel H: n = 7 mice per group.


B - C , Changes in lipid content ( total amount ) observed in THEM6 KO CRPC when compared to CTL . Data information : Data are presented as mean values + / - SD . Statistical : * p - value < 0 . 05 using a 1 - way ANOVA with a Dunnett ' s multiple comparisons . Data reproducibility : n = 3 independent biological experiments . DAG : diacylglycerol ; TG : triglyceride ; PC : phosphatidylcholine ; PE : phosphatidylethanolamine ; PI : phosphatidylinositol ; LysoPC : lysophosphatidylcholine ; LysoPE : lysophosphatidylethanolamine ; Cer : Ceramide ; SM : sphingomyelin ; CE : Cholesteryl ester .

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B-C, Changes in lipid content (total amount) observed in THEM6 KO CRPC cells when compared to CTL. Data information: Data are presented as mean values +/- SD. Statistical analysis : *p-value < 0.05 using a 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent biological experiments. DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester.


C , Representative electron microscopy ( EM ) pictures of CTL THEM6 KO 22rv1 taken at low ( left ) high ( right ) magnification . Red arrows point towards abnormal ER structure . Scale bar represents 500 nm .

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C, Representative electron microscopy (EM) pictures of CTL and THEM6 KO 22rv1 cells taken at low (left) and high (right) magnification. Red arrows point towards abnormal ER structure. Scale bar represents 500 nm.


I , Western blot of THEM6 , AMFR , SEC61b XPO1 expression in CRPC following THEM6 immunoprecipitation in CRPC . Data information : Data reproducibility : representative image from 2 independent biological experiments .

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I, Western blot analysis of THEM6, AMFR, SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells. Data information: Data reproducibility: representative image from 2 independent biological experiments.


J , Western blot of CALX CALR expression in PCa following THEM6 silencing . Data information : Data reproducibility : representative image from 3 independent biological experiments .

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J, Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing. Data information: Data reproducibility: representative image from 3 independent biological experiments.


B , RT - qPCR of MVD , FDPS DHCR7 expression in CTL THEM6 KO 22rv1 . CASC3 was used as a normalising control . Data information : Data are presented as mean values + / - SD . Statistical : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons . Data reproducibility : : n = 3 independent biological experiments .

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B, RT-qPCR analysis of MVD, FDPS and DHCR7 expression in CTL and THEM6 KO 22rv1 cells. CASC3 was used as a normalising control. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: : n = 3 independent biological experiments.


F , Labelled cholesterol fraction derived from 13C - glucose 13C - glutamine in CTL THEM6 KO CWR22res after 72 hours of incubation . G , Labelled cholesterol fraction derived from 13C - glucose 13C - glutamine in CTL THEM6 KO MCF - 7 after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical : , F , : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons . G : * p - value < 0 . 05 using a two - Student t - . Data reproducibility : : n = 3 independent wells from same culture .

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F, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO CWR22res cells after 72 hours of incubation. G, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO MCF-7 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: , F, : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. G: *p-value < 0.05 using a two-tailed Student t-test. Data reproducibility: : n = 3 independent wells from the same cell culture.


J , Labelled palmitic , oleic stearic acid fractions derived from 13C - glucose 13C - glutamine in CTL THEM6 KO 22rv1 after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons . Data reproducibility : n = 3 independent wells from same culture .

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J, Labelled palmitic, oleic and stearic acid fractions derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent wells from the same cell culture.


B , Proteomic highlighting associated with ER stress response significantly down - regulated in THEM6 KO 22rv1 when compared to CTL ( p - value ≤ 0 . 05 , FC = 1 . 3 ) . Data information : Data are presented as mean values + / - SD . Data reproducibility : n = 3 independent biological experiments .

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B, Proteomic analysis highlighting proteins associated with the ER stress response significantly down-regulated in THEM6 KO 22rv1 cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.


E , Proteomic highlighting ATF4 targets significantly down - regulated in THEM6 KO LNCaP AI when compared to CTL ( p - value ≤ 0 . 05 , FC = 1 . 3 ) . Data information : Data are presented as mean values + / - SD . Data reproducibility : n = 3 independent biological experiments .

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E, Proteomic analysis highlighting ATF4 targets significantly down-regulated in THEM6 KO LNCaP AI cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.


J , Western blot of ATF4 CHOP expression in CTL THEM6 KO LNCaP AI treated with palmitic acid ( 200 µM ) or hexadecylglycerol ( 50 µM ) for 48 hours . Data information : HSC70 was used as a sample loading control . Data reproducibility : representative image from 3 independent biological experiments .

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J, Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid (200 µM) or hexadecylglycerol (50 µM) for 48 hours. Data information: HSC70 was used as a sample loading control. Data reproducibility: representative image from 3 independent biological experiments.


C , Kaplan - Meier progression - free survival of PCa stratified according high low THEM6 expression using PRAD TCGA dataset . D , Kaplan - Meier recurrence - free survival of PCa stratified according to median THEM6 expression using GSE21034 dataset ( n = 179 ) . Data information : Statistical : logrank . Data reproducibility : C : n = 489 tumours . D : n = 179 .

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C, Kaplan-Meier progression-free survival analysis of PCa patients stratified according high and low THEM6 expression using the PRAD TCGA dataset. D, Kaplan-Meier recurrence-free survival analysis of PCa patients stratified according to median THEM6 expression using the GSE21034 dataset (n = 179). Data information: Statistical analysis: logrank test. Data reproducibility: C: n = 489 tumours. D: n = 179.


E , of THEM6 expression in treatment naïve , NHT - treated , CRPC NEPC tumours . Scale bar represents 100 µm . Data information : Data reproducibility : : n = 132 ; 90 ; 66 ; 137 ; 44 ; 30 tumours for Untreated ; NHT 1 - 3 ; NHT4 - 6 ; NHT > 7 ; CRPC ; NEPC respectively .

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E, IHC staining of THEM6 expression in treatment naïve, NHT-treated, CRPC and NEPC tumours. Scale bar represents 100 µm. Data information: Data reproducibility: : n = 132; 90; 66; 137; 44; 30 tumours for Untreated; NHT 1-3; NHT4-6; NHT>7; CRPC; NEPC respectively.


I - J , Kaplan - Meier recurrence - free ( I ) overall ( J ) survival of PCa stratified according to median THEM6 expression . Data information : Statistical : logrank . Data reproducibility : n = 35 tumours for THEM6 low n = 34 tumours for THEM6 high .

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I-J, Kaplan-Meier recurrence-free (I) and overall (J) survival analysis of PCa patients stratified according to median THEM6 expression. Data information: Statistical analysis: logrank test. Data reproducibility: n = 35 tumours for THEM6 low and n = 34 tumours for THEM6 high.


K , of THEM6 expression in PCa tissue samples according to Ki67 expression in analysed in F . L , of THEM6 expression in PCa tissue samples according to Ki67 expression in analysed in I - J . Data information : Centre corresponds to median of data , top bottom of box correspond to 90th 10th percentile , respectively . Whiskers extend to adjacent values ( minimum maximum data points not considered outliers ) . Statistical : two - Mann - Whitney U . Data reproducibility : K : n = 269 tumours for Ki67 low n = 270 tumours for Ki67 high . L : n = 35 tumours for Ki67 low n = 29 tumours for Ki67 high .

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K, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in F. L, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in I-J. Data information: Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: two-tailed Mann-Whitney U test. Data reproducibility: K: n = 269 tumours for Ki67 low and n = 270 tumours for Ki67 high. L: n = 35 tumours for Ki67 low and n = 29 tumours for Ki67 high.


M , Volcano plot of differentially modulated genes in PCa from PRAD TCGA stratified according to THEM6 expression . Red dots represent UPR - related genes extracted from UPR - gene signature Data information : Data reproducibility : n = 489 tumours .

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M, Volcano plot of the differentially modulated genes in PCa patients from the PRAD TCGA stratified according to THEM6 expression. Red dots represent the UPR-related genes extracted from the UPR-gene signature Data information: Data reproducibility: n = 489 tumours.


( A ) Atg5fl / fl Cre − Atg5fl / fl Cre + bone marrow ‐ derived macrophages ( BMMs ) , pretreated overnight with 100 ng / ml LPS , were stimulated for 1 h with inflammasome agonist nigericin ( 20 μM ) with ( Starvation ; EBSS ) or without ( Full ; full medium ) autophagic induction . culture supernatants were assayed for murine IL ‐ 1β by ELISA . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 .

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(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n≥3); *P0.05.


( B ) LPS ‐ pretreated Atg5fl / fl Cre − Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM release of active caspase ‐ 1 IL ‐ 1β was determined by immunoblotting .

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(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase‐1 and IL‐1β was determined by immunoblotting.


( C ) As in ( A ) , assayed for IL ‐ 18 . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 .

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(C) As in (A), assayed for IL‐18. Data represent mean values±s.d. (n≥3); *P0.05.


( D ) LPS ‐ pretreated BMMs were exposed to alum ( 250 μg / ml ) for 1 h with or without autophagic induction by starvation . Secreted IL ‐ 1β was measured as in ( A ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(D) LPS‐pretreated BMMs were exposed to alum (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.


( E ) LPS ‐ pretreated BMMs were exposed to silica ( 250 μg / ml ) for 1 h with or without autophagic induction by starvation . Secreted IL ‐ 1β was measured as in ( A ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

(E) LPS‐pretreated BMMs were exposed to silica (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.


( F ) BMMs were transfected with scramble ( Scr ) control siRNA or siRNAs against ASC NLRP3 . After 48 h following transfection , were treated overnight with LPS subjected to nigericin ( 20 μM ) starvation for 1 h . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(F) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to nigericin (20 μM) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.


( G ) Immunoblot of ASC NLRP3 knockdowns .

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(G) Immunoblot analysis of ASC and NLRP3 knockdowns.


( H ) BMMs were transfected with scramble ( Scr ) control siRNA or siRNAs against ASC NLRP3 . After 48 h following transfection , were treated overnight with LPS subjected to silica ( 250 μg / ml ) starvation for 1 h . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(H) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to silica (250 μg/ml) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.


( I ) Colocalization of IL ‐ 1β with basal autophagic machinery factor LC3 . : LC3 ( green , Alexa488 ) ; IL ‐ 1β ( red , Alexa568 ) . BMMs were from GFP - LC3 knock ‐ in mice , treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP IL ‐ 1β .

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(I) Colocalization of IL‐1β with the basal autophagic machinery factor LC3. Fluorescence: LC3 (green, Alexa488); IL‐1β (red, Alexa568). BMMs were from GFP-LC3 knock‐in mice, treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL‐1β.


(J, K) A line fluorescence tracing from images in (I).


( L ) Pearson ' s colocalization coefficient for IL ‐ 1β LC3 . Pearson ' s coefficient was derived from three independent experiments with five fields per experiment , for a total of 15 fields contributing to cumulative result . Figure source data can be found in Supplementary data .

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(L) Pearson's colocalization coefficient for IL‐1β and LC3. Pearson's coefficient was derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.


( A , B ) LPS ‐ pretreated BMMs were treated with 20 μM nigericin ( Nig ) 100 nM bafilomycin A1 ( Baf ) with ( Starvation ) or without ( Full ) autophagic induction for 1 h secreted IL ‐ 1β ( A ) IL ‐ 18 ( B ) were measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A, B) LPS‐pretreated BMMs were treated with 20 μM nigericin (Nig) and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β (A) and IL‐18 (B) were measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( C ) LPS ‐ pretreated BMMs were treated with 250 μg / ml of silica 100 nM bafilomycin A1 ( Baf ) with ( Starvation ) or without ( Full ) autophagic induction for 1 h secreted IL ‐ 1β were measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(C) LPS‐pretreated BMMs were treated with 250 μg/ml of silica and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β were measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( D ) Colocalization of cathepsin B with basal autophagic machinery factor LC3 IL ‐ 1β . ; LC3 ( green , Alexa488 ) , IL ‐ 1β ( red , Alexa568 ) , cathepsin B ( blue , Alexa633 ) . BMMs from GFP - LC3 knock ‐ in mice were treated with LPS then analysed for immunofluorescence .

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(D) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), and cathepsin B (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were treated with LPS and then analysed for immunofluorescence.


( E ) Colocalization tracing from in ( D ) .

(E) Colocalization line tracing analysis from images in (D).


( F ) LPS ‐ pretreated BMMs were treated with 20 μM nigericin cathepsin B inhibitor CA ‐ 074 Me ( 10 μM ) , with ( Starvation ) or without ( Full ) autophagic induction , for 1 h secreted IL ‐ 1β was measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(F) LPS‐pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA‐074 Me (10 μM), with (Starvation) or without (Full) autophagic induction, for 1 h and secreted IL‐1β was measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( G ) LPS ‐ pretreated Atg5fl / fl Cre − Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM release of cathepsin B was determined by immunoblotting . Figure source data can be found in Supplementary data .

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(G) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting. Figure source data can be found in Supplementary data.


( A ) Colocalization of Rab8a with basal autophagic machinery factor LC3 IL ‐ 1β . ; LC3 ( green , Alexa488 ) , IL ‐ 1β ( red , Alexa568 ) , Rab8a ( blue , Alexa633 ) . BMMs from GFP - LC3 knock ‐ in mice were pretreated with LPS analysed by immunofluorescence microscopy . Arrows indicate triple colocalization .

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(A) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization.


(B) Line tracing analysis of fluorescence signal intensity.


( C ) Pearson ' s colocalization coefficient for IL ‐ 1β Rab8a . Pearson ' s coefficients were derived from three completely independent experiments with > 5 fields per experiment , for a total of ⩾ 15 fields contributing to cumulative result .

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(C) Pearson's colocalization coefficient for IL‐1β and Rab8a. Pearson's coefficients were derived from three completely independent experiments with >5 fields per experiment, for a total of ⩾15 fields contributing to the cumulative result.


( D ) BMMs were transfected with siRNAs against Rab8a or scramble ( Scr ) control . At 24 h after first transfection , were transfected again with siRNA , treated with LPS day after subjected to nigericin in full medium for 1 h , IL ‐ 1β secretion measured .

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(D) BMMs were transfected with siRNAs against Rab8a or scramble (Scr) control. At 24 h after the first transfection, cells were transfected again with siRNA, treated with LPS and the day after subjected to nigericin in full medium for 1 h, and IL‐1β secretion measured.


( E ) Immunoblot of Rab8a knockdown in BMMs .

(E) Immunoblot analysis of Rab8a knockdown in BMMs.


( F ) RAW 264 . 7 macrophages were transfected with GFP ‐ tagged Rab8a constructs ( WT , wild type ; S22N , dominant ‐ negative mutant ) , treated overnight with LPS stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation . IL ‐ 1β secretion was measured by ELISA . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 . Figure source data can be found in Supplementary data .

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(F) RAW 264.7 macrophages were transfected with GFP‐tagged Rab8a constructs (WT, wild type; S22N, dominant‐negative mutant), treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation. IL‐1β secretion was measured by ELISA. Data represent mean values±s.d. (n≥3); *P0.05. Figure source data can be found in Supplementary data.


( A ) BMM were transfected with scramble ( Scr ) control siRNA or siRNA against GRASP55 . After 48 h of transfection , were treated with LPS day after subjected to 20 μM nigericin in EBSS , secreted IL ‐ 1β was measured by ELISA . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 . Inset : Immunoblot of GRASP55 knockdown .

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(A) BMM cells were transfected with scramble (Scr) control siRNA or siRNA against GRASP55. After 48 h of transfection, cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS, and secreted IL‐1β was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Inset: Immunoblot analysis of GRASP55 knockdown.


( B ) Immunofluorescence confocal microscopy of LC3 GRASP55 distribution . LC3 ( green , Alexa488 ) , GRASP55 ( red , Alexa568 ) . BMMs were pretreated overnight with 100 ng / ml LPS either not stimulated ( Ctrl ) or stimulated ( Nig ) for 30 min with inflammasome agonist nigericin ( 20 μM ) in full medium .

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(B) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution. LC3 (green, Alexa488), GRASP55 (red, Alexa568). BMMs were pretreated overnight with 100 ng/ml LPS and either not stimulated (Ctrl) or stimulated (Nig) for 30 min with the inflammasome agonist nigericin (20 μM) in full medium.


( C ) tracings , of signal from in ( B ) .

(C) Line tracings, analysis of fluorescence signal intensity from images in (B).


( D ) Pearson ' s coefficients for LC3 GRASP55 were quantified using SlideBook morphometric software as a measure of adjacency between GRASP55 LC3 profiles . Pearson ' s coefficients were derived from three independent experiments with five fields per experiment , for a total of 15 fields contributing to cumulative result . Figure source data can be found in Supplementary data .

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(D) Pearson's coefficients for LC3 and GRASP55 were quantified using SlideBook morphometric analysis software as a measure of adjacency between GRASP55 and LC3 profiles. Pearson's coefficients were derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.


( A , B ) Effect of GRASP55 on autophagy induction by measuring LC3 ‐ II . BMM were transfected with GRASP55 siRNAs or scramble ( Scr ) control . At 72 h post transfection , were induced for autophagy , treated or not with Bafilomycin A1 ( Baf ) to inhibit autophagic degradation LC3 ‐ II / actin ratios determined by immunoblotting ( A ) followed by densitometry ( B ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P0.05.


( C , D ) RAW 264 . 7 was transfected with GRASP55 siRNAs or scramble ( Scr ) siRNA control . Following 48 h of siRNA treatment , were transfected with RFP - GFP - LC3 plasmid ( GFP is sensitive to acidification , whereas RFP is not ) , after 24 h induced for autophagy in EBSS for 1 h autophagic induction flux quantified ( graph in D ) by determining number of early autophagic organelles ( GFP + RFP + ) autolysosomal organelles ( GFP − RFP + ) per as illustrated in ( yellow arrows , GFP + RFP + ; red arrows , GFP − RFP + ) . Total , yellow + red per . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 . Figure source data can be found in Supplementary data .

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(C, D) RAW 264.7 was transfected with GRASP55 siRNAs or scramble (Scr) siRNA control. Following 48 h of siRNA treatment, cells were transfected with RFP-GFP-LC3 plasmid (GFP is sensitive to acidification, whereas RFP is not), after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified (graph in D) by determining the number of early autophagic organelles (GFP+RFP+ puncta) and autolysosomal organelles (GFP−RFP+ puncta) per cell as illustrated in fluorescent images (yellow arrows, GFP+RFP+; red arrows, GFP−RFP+). Total, yellow+red puncta per cell. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.


( A ) Atg5fl / fl Cre − Atg5fl / fl Cre + bone marrow ‐ derived macrophages ( BMMs ) , pretreated overnight with 100 ng / ml LPS , were stimulated for 1 h with inflammasome agonist nigericin ( 20 μM ) with ( Starvation ; EBSS ) or without ( Full ; full medium ) autophagic induction . culture supernatants were assayed for murine IL ‐ 1β by ELISA . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05.


( B ) LPS ‐ pretreated Atg5fl / fl Cre − Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM release of HMGB1 was determined by immunoblotting . Figure source data can be found in Supplementary data .

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(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.


A Coolness stimulates intracellular cyclic guanosine monophosphate ( cGMP ) accumulation in HEK ‐ 293T expressing GC ‐ G . Two days after transfection of empty vector or GC ‐ G ‐ encoding plasmid , were exposed to ambient temperatures ( 37 or 15 ° C ) for 20 min cGMP was measured .

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A Coolness stimulates intracellular cyclic guanosine monophosphate (cGMP) accumulation in HEK‐293T cells expressing GC‐G. Two days after transfection of empty vector or GC‐G‐encoding plasmid, cells were exposed to ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured.


B dependence of GC ‐ G activity . preparations from HEK ‐ 293T transfected with empty vector or GC ‐ G ‐ encoding plasmid were used to determine cyclase activity at indicated temperatures for 20 min ( asterisks denote values significantly elevated compared to 37 ° C ) .

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B Temperature dependence of GC‐G activity. Membrane preparations from HEK‐293T cells transfected with empty vector or GC‐G‐encoding plasmid were used to determine cyclase activity at the indicated temperatures for 20 min (asterisks denote values significantly elevated compared to 37°C).


C GC ‐ G is only murine transmembrane GC that can be stimulated by cool temperatures . Expression plasmids encoding FLAG ‐ tagged GC subtypes GC ‐ A to GC ‐ G were transfected into HEK ‐ 293T . Two days after transfection , fractions were prepared used for cyclase activity assays at 37 or 15 ° C . expression of each receptor GC was confirmed by Western blot ( WB ) with anti ‐ FLAG antibody .

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C GC‐G is the only murine transmembrane GC that can be stimulated by cool temperatures. Expression plasmids encoding FLAG‐tagged GC subtypes GC‐A to GC‐G were transfected into HEK‐293T cells. Two days after transfection, membrane protein fractions were prepared and used for cyclase activity assays at 37 or 15°C. Protein expression of each receptor GC was confirmed by Western blot (WB) analysis with anti‐FLAG antibody.


D H + CYC domain is critical for stimulation by coolness . domain structure of full ‐ ( FL ) GC ‐ G its mutant variants is shown in upper panel . A FLAG tag was added at N ‐ terminus of each . ΔECD + KLD mutant lacks 73 - 455 556 - 833 ; ΔCYC variant lacks 834 - 1 , 003 . H + CYC mutant contains 806 - 1 , 100 . ECD , extracellular domain ; KLD , kinase ‐ like domain ; SP , signal peptide ; TM , transmembrane region . HEK ‐ 293T expressing indicated GC ‐ G constructs were incubated at 37 or 15 ° C for 20 min , then intracellular cGMP was measured ( lower panel ) .

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D The H+CYC domain is critical for stimulation by coolness. The domain structure of full‐length (FL) GC‐G and its mutant variants is shown in the upper panel. A FLAG tag was added at the N‐terminus of each protein. The ΔECD+KLD mutant protein lacks amino acids 73-455 and 556-833; the ΔCYC variant lacks amino acids 834-1,003. The H+CYC mutant protein contains amino acids 806-1,100. ECD, extracellular domain; KLD, kinase‐like domain; SP, signal peptide; TM, transmembrane region. HEK‐293T cells expressing the indicated GC‐G constructs were incubated at 37 or 15°C for 20 min, then intracellular cGMP concentration was measured (lower panel).


In chimeric GC ‐ A / G , H + CYC domain of GC ‐ A was replaced by that of GC ‐ G ( upper panel ) . HEK ‐ 293T expressing indicated were exposed to ambient of 37 or 15 ° C for 20 min ; then , intracellular cGMP was measured . Data are mean ± SD from three experiments in triplicate . * * P 0 . 01 .

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In the chimeric GC‐A/G protein, the H+CYC domain of GC‐A was replaced by that of GC‐G (upper panel). HEK‐293T cells expressing the indicated proteins were exposed to ambient temperature of 37 or 15°C for 20 min; then, intracellular cGMP concentration was measured. Data are mean ± SD from three experiments in triplicate. **P 0.01.


B , C expression coolness ‐ stimulated cGMP synthesis of above shown divergent C ‐ terminal fragments of GC ‐ G with intact or ablated hinge ( H ) region . expression plasmids encoding H + CYC domain or a deletion construct ( Δ1 - Δ2 ) tagged with a FLAG epitope were transfected into HEK ‐ 293T . Two days after transfection , were exposed to ambient temperatures ( 37 or 15 ° C ) for 20 min cGMP was measured ( C ) . expression of each construct was confirmed by Western blot ( WB ) with anti ‐ FLAG antibody ( B ) .

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B, C Protein expression and coolness‐stimulated cGMP synthesis of the above shown divergent C‐terminal fragments of GC‐G with intact or ablated hinge (H) region. The expression plasmids encoding the H+CYC domain or a deletion construct (Δ1-Δ2) tagged with a FLAG epitope were transfected into HEK‐293T cells. Two days after transfection, cells were exposed to the ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured (C). Protein expression of each construct was confirmed by Western blot (WB) analysis with anti‐FLAG antibody (B).


D A recombinant containing H + CYC domain of GC ‐ G acquires coolness ‐ evoked activity . Diagram of recombinant GST . GC ‐ G ‐ CYC containing H + CYC domain of GC ‐ G ( 849 - 1 , 078 ) compared to domain structure of full ‐ GC ‐ G ( upper panel ) . purified GST . GC ‐ G ‐ CYC was exposed to indicated temperatures for 20 min , GST . GC ‐ G ‐ CYC cyclase activity was measured ( lower panel ; asterisks denote values significantly elevated compared to 37 ° C ) .

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D A recombinant protein containing the H+CYC domain of GC‐G acquires coolness‐evoked activity. Diagram of recombinant GST.GC‐G‐CYC protein containing the H+CYC domain of GC‐G (amino acids 849-1,078) compared to the domain structure of full‐length GC‐G (upper panel). The purified GST.GC‐G‐CYC protein was exposed to the indicated temperatures for 20 min, and GST.GC‐G‐CYC cyclase activity was measured (lower panel; asterisks denote values significantly elevated compared to 37°C).


A HEK ‐ 293T were co ‐ transfected with plasmids encoding two differential epitope ‐ tagged ( FLAG or Myc ) versions of GC ‐ G or GC ‐ A , respectively . After 2 days , transfected were lysed with detergent lysates were subjected to immunoprecipitation ( IP ) for 2 h with anti ‐ Myc antibody at two different temperatures ( 15 or 25 ° C ) , followed by Western blotting ( WB ) using anti ‐ FLAG antibody to determine amount of dimerized / oligomerized GC ( upper panel ) . These approaches demonstrated that in contrast to GC ‐ A , homomeric / association of GC ‐ G seems to be enhanced by cool temperatures ( upper panel ) . lysates were also precipitated immunoblotted with anti ‐ Myc antibody , demonstrating that cooling does not interfere with immunoprecipitation of Myc ‐ tagged GC ‐ G via anti ‐ Myc antibody ( middle panel ) . Likewise , precipitation immunoblotting with anti ‐ FLAG antibody yielded bands of similar for 15 25 ° C , respectively ( lower panel ) . B Relative of homomeric / association of GC ‐ G GC ‐ A was performed by densitometric scanning . Each value from upper panel was subsequently normalized to total as determined in middle lower panel of Fig A ( of immunoreactive bands obtained with anti ‐ Myc or anti ‐ FLAG antibody , respectively ) . Relative dimerization level at 15 ° C was further calculated by normalizing to values obtained at 25 ° C ; latter were set as 1 . Results are means ± SD of three experiments , * * P 0 . 01 ( compared to 25 ° C ) .

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

A HEK‐293T cells were co‐transfected with plasmids encoding two differential epitope‐tagged (FLAG or Myc) versions of GC‐G or GC‐A, respectively. After 2 days, transfected cells were lysed with detergent and lysates were subjected to immunoprecipitation (IP) for 2 h with anti‐Myc antibody at two different temperatures (15 or 25°C), followed by Western blotting (WB) using anti‐FLAG antibody to determine the amount of dimerized/oligomerized GC proteins (upper panel). These approaches demonstrated that in contrast to GC‐A, homomeric protein/protein association of GC‐G seems to be enhanced by cool temperatures (upper panel). Cell lysates were also precipitated and immunoblotted with the anti‐Myc antibody, demonstrating that cooling does not interfere with immunoprecipitation of Myc‐tagged GC‐G via the anti‐Myc antibody (middle panel). Likewise, precipitation and immunoblotting with the anti‐FLAG antibody yielded bands of similar intensity for 15 and 25°C, respectively (lower panel).B Relative quantification of homomeric protein/protein association of GC‐G and GC‐A was performed by densitometric scanning. Each intensity value from the upper panel was subsequently normalized to the total protein levels as determined in the middle and the lower panel of Fig A (intensity of the immunoreactive bands obtained with the anti‐Myc or anti‐FLAG antibody, respectively). Relative dimerization level at 15°C was further calculated by normalizing to values obtained at 25°C; the latter were set as 1. Results are means ± SD of three experiments, **P 0.01 (compared to 25°C).


HEK ‐ 293T expressing CNGA3 alone ( upper panel ) or together with GC ‐ G ( full ‐ in middle panel H + CYC domain in lower panel ) were loaded with Ca2 + indicator Fura ‐ 2 to monitor intracellular [ Ca2 + ] i . co ‐ expressing GC ‐ G CNGA3 showed a rapid increase in [ Ca2 + ] i when was lowered from 37 to 15 ° C ( middle lower panel ) ; only expressing CNGA3 are not responsive to cool temperatures ( top panel ) . Removal of extracellular Ca2 + ( − Ca2 + ; middle panel ) completely suppressed response to cooling . ΔF represents changes in of of Fura ‐ 2 at 340 / 380 nm excitation . ratiometric Ca2 + responses are representative of 30 recorded from three experiments .

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

HEK‐293T cells expressing CNGA3 alone (upper panel) or together with GC‐G (full‐length in the middle panel and H+CYC domain in the lower panel) were loaded with the Ca2+ indicator Fura‐2 to monitor intracellular [Ca2+]i concentration. Cells co‐expressing GC‐G and CNGA3 showed a rapid increase in [Ca2+]i when the temperature was lowered from 37 to 15°C (middle and lower panel); cells only expressing CNGA3 are not responsive to cool temperatures (top panel). Removal of extracellular Ca2+ (−Ca2+; middle panel) completely suppressed the response to cooling. ΔF represents changes in the ratio of the fluorescence intensity of Fura‐2 at 340/380 nm excitation. The ratiometric Ca2+ responses are representative of 30 cells recorded from three experiments.


A - H In situ hybridization experiments with an antisense probe for c ‐ Fos on coronal sections through GG of wild ‐ type ( WT ; left panel ) or GC ‐ G ‐ KO ( right panel ) mouse pups ( P0 - P4 ) exposed to warm ( 35 ° C ; A - D ) or cool ( 22 ° C ; E - H ) ambient for 2 h . Panels ( C , D , G , H ) show higher magnifications of boxed areas in ( A , B , E , F ) . At 35 ° C ( A - D ) , no c ‐ Fos expression was detectable in GG of both WT ( A , C ) GC ‐ G ‐ KO ( B , D ) individuals . Upon exposure to 22 ° C , strong c ‐ Fos expression was observed in GG of WT pups ( E , G ) , whereas c ‐ Fos expression was barely detectable in GG of GC ‐ G ‐ KO ( F , H ) . data shown are representative of 10 independent experiments . Pups originated from six different litters for each genotype . Scale bars : 200 μm in ( A , B , E , F ) ; 50 μm in ( C , D , G , H ) . I of c ‐ Fos ‐ positive GG in WT GC ‐ G ‐ KO pups on exposure to 22 ° C for 2 h . stained on every section along rostrocaudal extent of GG were counted ; results shown are on above ‐ mentioned 10 experiments . number of c ‐ Fos ‐ positive in GG of a GC ‐ G ‐ KO mouse was determined relative to that in a concomitantly processed WT pup ; latter number was set to 100 % . Data are mean ± SD ( n = 11 in each genotype ) , * * P 0 . 0001 .

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

A-H In situ hybridization experiments with an antisense probe for c‐Fos on coronal sections through the GG of wild‐type (WT; left panel) or GC‐G‐KO (right panel) mouse pups (P0-P4) exposed to warm (35°C; A-D) or cool (22°C; E-H) ambient temperature for 2 h. Panels (C, D, G, H) show higher magnifications of the boxed areas in (A, B, E, F). At 35°C (A-D), no c‐Fos expression was detectable in the GG of both WT (A, C) and GC‐G‐KO (B, D) individuals. Upon exposure to 22°C, strong c‐Fos expression was observed in the GG of WT pups (E, G), whereas c‐Fos expression was barely detectable in the GG of GC‐G‐KO animals (F, H). The data shown are representative of 10 independent experiments. Pups originated from six different litters for each genotype. Scale bars: 200 μm in (A, B, E, F); 50 μm in (C, D, G, H).I Quantification of c‐Fos‐positive GG cells in WT and GC‐G‐KO pups on exposure to 22°C for 2 h. All stained cells on every section along the rostrocaudal extent of the GG were counted; the results shown are based on the above‐mentioned 10 experiments. The number of c‐Fos‐positive cells in the GG of a GC‐G‐KO mouse was determined relative to that in a concomitantly processed WT pup; the latter number was set to 100%. Data are mean ± SD (n = 11 in each genotype), **P 0.0001.


J - N Calcium imaging of coronal sections through GG of olfactory marker ‐ green ( OMP ‐ GFP ) / GC ‐ G + / + or OMP ‐ GFP / GC ‐ G ‐ KO pups ( P1 - P4 ) . High magnification image ( J ) of a tissue slice through GG of an OMP ‐ GFP pup with GG neurons labeled by intrinsic GFP ( GFP was merged with transmitted ‐ channel ) . analyzed are circled in blue ( GFP ‐ negative ) or red ( GFP ‐ positive ) . NC , nasal cavity . Scale bar : 30 μm . ( K - M ) Representative ratiometric Ca2 + transients induced by cooling from 37 to 15 ° C in GFP ‐ negative non ‐ neuronal ( K ) in GFP ‐ positive GG neurons from OMP ‐ GFP / GC ‐ G + / + ( L ) OMP ‐ GFP / GC ‐ G ‐ KO pups ( M ) . numbers in bottom right corners are number of with Ca2 + transient similar to what is shown in respective graph ( left ) total number of measured ( right ) . ( N ) of coolness ‐ induced ΔF in GG neurons from OMP ‐ GFP / GC ‐ G + / + OMP ‐ GFP / GC ‐ G ‐ KO mice . Coolness ‐ induced ΔF was calculated by subtracting baseline ( 340 / 380 nm ) at 37 ° C from peak ( 340 / 380 nm ) measured at 15 ° C . For OMP ‐ GFP / GC ‐ G + / + , 29 coolness ‐ responsive neurons ( black bar ) of 54 analyzed neurons ( gray bar ) from five slices ( obtained from different ) were analyzed . For OMP ‐ GFP / GC ‐ G ‐ KO , 50 analyzed neurons ( shaded bar ) from five slices ( obtained from different ) were analyzed . Data are mean ± SD , * * P 0 . 01 .

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

J-N Calcium imaging of coronal sections through the GG of olfactory marker protein‐green fluorescent protein (OMP‐GFP)/GC‐G+/+ or OMP‐GFP/GC‐G‐KO pups (P1-P4). High magnification image (J) of a tissue slice through the GG of an OMP‐GFP pup with GG neurons labeled by intrinsic GFP fluorescence (GFP fluorescence was merged with the transmitted‐light channel). Cells analyzed are circled in blue (GFP‐negative) or red (GFP‐positive). NC, nasal cavity. Scale bar: 30 μm. (K-M) Representative ratiometric Ca2+ transients induced by cooling from 37 to 15°C in GFP‐negative non‐neuronal cells (K) and in GFP‐positive GG neurons from OMP‐GFP/GC‐G+/+ (L) and OMP‐GFP/GC‐G‐KO pups (M). The numbers in the bottom right hand corners are the number of cells with Ca2+ transient similar to what is shown in the respective graph (left) and total number of measured cells (right). (N) Quantification of coolness‐induced ΔF in GG neurons from OMP‐GFP/GC‐G+/+ and OMP‐GFP/GC‐G‐KO mice. Coolness‐induced ΔF was calculated by subtracting the baseline fluorescence ratio (340/380 nm) at 37°C from the peak fluorescence ratio (340/380 nm) measured at 15°C. For OMP‐GFP/GC‐G+/+, the 29 coolness‐responsive neurons (black bar) of all 54 analyzed neurons (gray bar) from five slices (obtained from different animals) were analyzed. For OMP‐GFP/GC‐G‐KO, the 50 analyzed neurons (shaded bar) from five slices (obtained from different animals) were analyzed. Data are mean ± SD, **P 0.01.


A Reduced responsive rate of coolness ‐ stimulated USV in GC ‐ G ‐ KO pups . Pups were removed from their dams at postnatal ( P ) day 2 , 4 , 6 exposed to 15 ° C . USV was measured by use of Anabat SD1 Bat detector . number of responsive pups versus total number of pups tested for each WT GC ‐ G ‐ KO group at different ages is indicated on top of each bar . * P 0 . 05 . B , C Total number of USV calls ( B ) response latency ( C ) in GC ‐ G ‐ KO WT pups at P2 . horizontal bars indicate mean . * P 0 . 05 ; * * P 0 . 01 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O 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 Reduced responsive rate of coolness‐stimulated USV in GC‐G‐KO pups. Pups were removed from their littermates and dams at postnatal (P) day 2, 4, and 6 and exposed to 15°C. USV was measured by use of the Anabat SD1 Bat detector. The number of responsive pups versus total number of pups tested for each WT and GC‐G‐KO group at different ages is indicated on the top of each bar. *P 0.05.B, C Total number of USV calls (B) and response latency (C) in GC‐G‐KO and WT pups at P2. The horizontal bars indicate the mean. *P 0.05; **P 0.01.


Western blot for p53 , phosphorylated p53 at Ser15 ( p53 pS15 ) , vinculin ( vinc ) in HCT116 treated with 300 μM 5 - FU for 8 h silenced for PHD1 , PHD2 , or PHD3 .

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

Western blot for p53, phosphorylated p53 at Ser15 (p53 pS15), and vinculin (vinc) in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1, PHD2, or PHD3.


qRT - PCR for PHD1 , PHD2 , PHD3 in HCT116 silenced for PHD1 ( * P = 0 . 0006 toward Scr ) , PHD2 ( * P = 0 . 0004 toward Scr ) , or PHD3 ( * P = 0 . 0075 toward Scr ) . A two - unpaired t - was performed with n = 3 / group .

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

qRT-PCR for PHD1, PHD2, and PHD3 in HCT116 silenced for PHD1 (*P = 0.0006 toward Scr), PHD2 (*P = 0.0004 toward Scr), or PHD3 (*P = 0.0075 toward Scr). A two-tailed unpaired t-test was performed with n = 3/group.


Western blot of p53 pS15 , p53 , PHD1 , vinculin in HCT116 treated with 300 μM 5 - FU for 8 h silenced for PHD1 or a scrambled ( Scr ) control .

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

Western blot of p53 pS15, p53, PHD1, and vinculin in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1 or a scrambled (Scr) control.


qRT - PCR for PHD1 showing silencing efficacy with both siRNAs 1 ( * P = 0 . 0017 toward Scr ) 2 ( * P = 0 . 0023 toward Scr ) compared to scrambled control in HCT116 . A two - unpaired t - was performed with n = 3 / group .

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

qRT-PCR for PHD1 showing silencing efficacy with both siRNAs 1 (*P = 0.0017 toward Scr) and 2 (*P = 0.0023 toward Scr) compared to the scrambled control in HCT116. A two-tailed unpaired t-test was performed with n = 3/group.


Western blot for p53 , p53 pS15 , vinc in HCT116 silenced for PHD1 with two different constructs ( constructs 1 2 ) upon exposure to 300 μM 5 - FU for 8 h .

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

Western blot for p53, p53 pS15, and vinc in HCT116 silenced for PHD1 with two different constructs (constructs 1 and 2) upon exposure to 300 μM 5-FU for 8 h.


Western blot for p53 pS15 , p53 , vinculin in HCT116 silenced for PHD1 treated with either 200 nM SN - 38 or 20 μM oxaliplatin for 8 h .

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

Western blot for p53 pS15, p53, and vinculin in HCT116 silenced for PHD1 and treated with either 200 nM SN-38 or 20 μM oxaliplatin for 8 h.


of PHD1 in LIM1215 silenced for PHD1 . ( * P = 0 . 0099 toward Scr in a two - unpaired t - with n = 3 / group . )

O B-MEASURED_VAR 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

RNA levels of PHD1 in LIM1215 silenced for PHD1. (*P = 0.0099 toward Scr in a two-tailed unpaired t-test with n = 3/group.)


Western blot for p53 pS15 , p53 , vinc in LIM1215 upon silencing of PHD1 treatment for 8 h with 200 μM 5 - FU .

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

Western blot for p53 pS15, p53, and vinc in LIM1215 upon silencing of PHD1 and treatment for 8 h with 200 μM 5-FU.


A Western blot for p53 , caspase - 3 ( casp3 ) , parp , vinculin ( vinc ) in p53wt / wt p53 − / − HCT116 silenced for PHD1 upon exposure to 300 μM 5 - FU for 24 h , showing an increased apoptotic response to 5 - FU treatment in PHD1 - silenced compared to scrambled control .

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

A Western blot for p53, cleaved caspase-3 (cleaved casp3), cleaved parp, and vinculin (vinc) in p53wt/wt and p53−/− HCT116 silenced for PHD1 upon exposure to 300 μM 5-FU for 24 h, showing an increased apoptotic response to 5-FU treatment in PHD1-silenced cells compared to the scrambled control.


B Confirmation of these results by FACS of propidium iodide - stained PHD1 - silenced p53wt / wt p53 − / − HCT116 exposed for 26 h to 300 - μM 5 - FU treatment . * P = 0 . 002 toward p53wt / wt HCT116 shScr 5 - FU - treated ( two - unpaired t - ) with n = 6 for non - treated p53wt / wt HCT116 n = 3 in other groups .

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

B Confirmation of these results by FACS analysis of propidium iodide-stained PHD1-silenced p53wt/wt and p53−/− HCT116 cells exposed for 26 h to 300-μM 5-FU treatment. *P = 0.002 toward p53wt/wt HCT116 shScr 5-FU-treated (two-tailed unpaired t-test) with n = 6 for non-treated p53wt/wt HCT116 and n = 3 in all other groups.


C Detection of casp3 , parp , vinc in HCT116 silenced with a second siRNA for PHD1 .

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

C Detection of cleaved casp3, cleaved parp, and vinc in HCT116 silenced with a second siRNA for PHD1.


D , E Apoptosis as detected by Western blot for casp3 parp in PHD1 - silenced HCT116 treated for 20 h with 200 nM SN - 38 ( D ) or for 24 h with 20 μM oxaliplatin ( E ) .

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D, E Apoptosis as detected by Western blot for cleaved casp3 and parp in PHD1-silenced HCT116 treated for 20 h with 200 nM SN-38 (D) or for 24 h with 20 μM oxaliplatin (E).


F Western blot for casp3 , parp , vinc in LIM1215 silenced for PHD1 treated for 24 h with 200 μM 5 - FU .

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

F Western blot for cleaved casp3, parp, and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5-FU.


G Similar apoptosis were detected by Western blot for casp3 parp with vinc as a loading control in p53S15A → p53 − / − HCT116 silenced for PHD1 or scrambled ( Scr ) control treated for 20 h with 300 μM 5 - FU .

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G Similar apoptosis levels were detected by Western blot for cleaved casp3 and parp with vinc as a loading control in p53S15A→p53−/− HCT116 silenced for PHD1 or scrambled (Scr) control and treated for 20 h with 300 μM 5-FU.


Representative of colony formation capacity of p53wt / wt HCT116 transduced with a doxycycline - inducible shScr or shPHD1 silencing construct , treated for 24 h with doxycycline with or without additional treatment for 8 h with 300 μM 5 - FU . * P = 0 . 008 toward shScr , two - unpaired t - n = 3 / group .

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

Representative images and quantification of the colony formation capacity of p53wt/wt HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct, treated for 24 h with doxycycline with or without additional treatment for 8 h with 300 μM 5-FU. *P = 0.008 toward shScr, two-tailed unpaired t-test and n = 3/group.


volume of p53wt / wtHCT116 transduced with a doxycycline - inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice treated with 5 - FU . * P = 0 . 045 toward shScr 5 - FU - treated mice by two - way ANOVA with n = 6 for p53wt / wtHCT116 shScr , n = 7 for p53wt / wtHCT116 shPHD1 p53wt / wtHCT116 shScr 5 - FU - treated , n = 8 for p53wt / wtHCT116 shPHD15 - FU - treated . volume of p53 − / − HCT116 transduced with a doxycycline - inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice treated with 5 - FU . n = 6 for p53 − / − HCT116 shScr , p53 − / − HCT116 shPHD1 , p53 − / − HCT116 shScr 5 - FU - treated n = 8 for p53 − / − HCT116 shPHD15 - FU - treated .

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

Tumor volume of p53wt/wtHCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. *P = 0.045 toward shScr 5-FU-treated mice by two-way ANOVA with n = 6 for p53wt/wtHCT116 shScr, n = 7 for p53wt/wtHCT116 shPHD1 and p53wt/wtHCT116 shScr 5-FU-treated, and n = 8 for p53wt/wtHCT116 shPHD15-FU-treated.Tumor volume of p53−/−HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. n = 6 for p53−/−HCT116 shScr, p53−/−HCT116 shPHD1, and p53−/−HCT116 shScr 5-FU-treated and n = 8 for p53−/−HCT116 shPHD15-FU-treated.


Western blot for p53 pS15 , p53 , vinculin ( vinc ) in PHD1 - silenced HCT116 upon treatment with 300 μM 5 - FU for 8 h with or without 0 . 5 mM DMOG for 26 h . Vinculin ( vinc ) is used as a loading control .

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

Western blot for p53 pS15, p53, and vinculin (vinc) in PHD1-silenced HCT116 upon treatment with 300 μM 5-FU for 8 h with or without 0.5 mM DMOG for 26 h. Vinculin (vinc) is used as a loading control.


Western blot detection of PHD1 - Flag p53 in whole extracts ( WCE ) or after immunoprecipitation against Flag - tag of overexpressed PHD1 - Flag in HEK293T .

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

Western blot detection of PHD1-Flag and p53 in whole cell extracts (WCE) or after immunoprecipitation against the Flag-tag of overexpressed PHD1-Flag in HEK293T cells.


Western blot detection of PHD1 - Flag p53 after immunoprecipitation against Flag - tag of recombinant PHD1 - Flag incubated with recombinant p53 .

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

Western blot detection of PHD1-Flag and p53 after immunoprecipitation against the Flag-tag of recombinant PHD1-Flag incubated with recombinant p53.


Western blot for p53 PHD1 in WCE or after immunoprecipitation of endogenous p53 in p53wt / wt HCT116 , silenced with a Scr control , siPHD1 , or sip53 , or after immunoprecipitation of p53wt / wt HCT116 lysates with an IgG control .

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

Western blot for p53 and PHD1 in WCE or after immunoprecipitation of endogenous p53 in p53wt/wt HCT116 cells, silenced with a Scr control, siPHD1, or sip53, or after immunoprecipitation of p53wt/wt HCT116 cell lysates with an IgG control.


Western blot for p53 pS15 , p53 , vinculin ( vinc ) in HCT116 treated with 300 μM 5 - FU upon silencing of PHD1 p38α , alone or in combination . Vinculin ( vinc ) is used as a loading control .

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

Western blot for p53 pS15, p53, and vinculin (vinc) in HCT116 treated with 300 μM 5-FU upon silencing of PHD1 and p38α, alone or in combination. Vinculin (vinc) is used as a loading control.


Detection of p53 pS15 p53 after in vitro phosphorylation by p38α , p38β , or p38γ of p53immunoprecipitated from silenced for a control ( SIMA ) or PHD1 .

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

Detection of p53 pS15 and p53 after in vitro phosphorylation by p38α, p38β, or p38γ of p53immunoprecipitated from cells silenced for a control (SIMA) or PHD1.


Detection by Western blot of p53 p38 from whole extracts ( WCE ) or after immunoprecipitation of p53 from silenced for a Scr control or siPHD1 treated for 1 h with 300 μM 5 - FU .

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

Detection by Western blot of p53 and p38 from whole cell extracts (WCE) or after immunoprecipitation of p53 from cell silenced for a Scr control or siPHD1 and treated for 1 h with 300 μM 5-FU.


Transcription of CDKN1A , GADD45 , MDM2 , BAX , PUMA in PHD1 - silenced p53wt / wt HCT116 either untreated or treated for 8 h with 300 μM 5 - FU .

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

Transcription analysis of CDKN1A, GADD45, MDM2, BAX, PUMA in PHD1-silenced p53wt/wt HCT116 either untreated or treated for 8 h with 300 μM 5-FU.


Western blot for parp vinculin ( vinc ) in PHD1 - silenced HCT116 treated for 20 h with 300 μM 5 - FU alone or in combination with 3 μg / ml α - amanitin .

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

Western blot for cleaved parp and vinculin (vinc) in PHD1-silenced HCT116 treated for 20 h with 300 μM 5-FU alone or in combination with 3 μg/ml α-amanitin.


PHD1 mRNA in p53R248 / − HCT116 silenced for PHD1 . * P = 0 . 0005 toward scrambled control with a two - unpaired t - n = 3 / group .

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

PHD1 mRNA levels in p53R248/− HCT116 silenced for PHD1. *P = 0.0005 toward scrambled control with a two-tailed unpaired t-test and n = 3/group.


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

Western blot for p53 pS15, p53, and vinculin in PHD1-silenced p53R248/− HCT116 upon 8-h treatment with 300 μM 5-FU.


Western blot for casp3 vinculin in p53R248 / − HCT116 silenced for PHD1 treated with 300 μM 5 - FU for 24 h .

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

Western blot for cleaved casp3 and vinculin in p53R248/− HCT116 silenced for PHD1 and treated with 300 μM 5-FU for 24 h.


Western blot for pH2AX vinculin ( vinc ) in p53wt / wt p53 − / − HCT116 after 24 - h treatment with 300 μM 5 - FU .

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

Western blot for pH2AX and vinculin (vinc) in p53wt/wt and p53−/− HCT116 cells after 24-h treatment with 300 μM 5-FU.


Detection of pH2AX vinc in p53S15A → p53 − / − HCT116 silenced for PHD1 treated with 300 μM 5 - FU for 24 h .

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

Detection of pH2AX and vinc in p53S15A→p53−/− HCT116 cells silenced for PHD1 and treated with 300 μM 5-FU for 24 h.


Detection of pH2AX vinc in HCT116 silenced with a second silencing construct of PHD1 treated for 24 h with 300 μM 5 - FU .

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

Detection of pH2AX and vinc in HCT116 silenced with a second silencing construct of PHD1 and treated for 24 h with 300 μM 5-FU.


Western blot for pH2AX vinc in HCT116 treated for 20 h with 200 nM SN - 38 or 24 h with 20 μM oxaliplatin .

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

Western blot for pH2AX and vinc in HCT116 treated for 20 h with 200 nM SN-38 or 24 h with 20 μM oxaliplatin.


Western blot for pH2AX vinc in p53R248 / − HCT116 silenced for PHD1 upon treatment with 300 μM 5 - FU for 24 h .

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

Western blot for pH2AX and vinc in p53R248/− HCT116 cells silenced for PHD1 upon treatment with 300 μM 5-FU for 24 h.


Detection by Western blot of p53 XPB from whole extracts ( WCE ) or after immunoprecipitation of p53 from silenced for Scr control or siPHD1 treated for 4 or 8 h with 300 μM 5 - FU .

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

Detection by Western blot of p53 and XPB from whole cell extracts (WCE) or after immunoprecipitation of p53 from cells silenced for Scr control or siPHD1 and treated for 4 or 8 h with 300 μM 5-FU.


Western blot for pH2AX vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5 - FU for 20 h .

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

Western blot for pH2AX and vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5-FU for 20 h.


Determination of serum testosterone ( B ) 17β - estradiol , in post - pubescent , castrated ( CtM ) , Hanwoo ( n = 5 in each group ) .

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

Determination of serum testosterone (B) and 17β-estradiol levels, in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


Determination of , in post - pubescent , castrated ( CtM ) , Hanwoo ( n = 5 in each group ) .

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

Determination of the body weight , in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


D . PCoA of rectal 16S rRNA gene sequences on weighted UniFrac matrix .

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

D. PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix.


E . Discriminant taxa of CtM rectal microbiota , as determined by linear discriminant effect ( LEfSe ) . different abundances are represented by LDA scores .

O O O 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. Discriminant taxa of the male and CtM rectal microbiota, as determined by the linear discriminant analysis effect size (LEfSe). The different abundances are represented by LDA scores.


F . serum metabolome profiles were analyzed using GC - TOF - MS clustered by PCoA on Bray - Curtis dissimilarity matrix .

F. The serum metabolome profiles were analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix.


G . relative abundances of serum metabolites displayed as bar graphs ( n = 5 in each group ) .

G. The relative abundances of serum metabolites displayed as bar graphs (n = 5 in each group).


H . BCAA serum quantified enzymatically , as described in materials methods section .

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

H. The BCAA serum levels quantified enzymatically, as described in the materials and methods section.


A . Serum testosterone in adult CtM Hanwoo ( n = 10 in each group ) . scattered dots in bar graph represent replicates .

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

A. Serum testosterone levels in the adult male and CtM Hanwoo (n = 10 in each group). The scattered dots in the bar graph represent the individual replicates.


PCoA , on weighted UniFrac matrix , of 16S rRNA gene data for rumen , ileum , colon , shown for different segments of gastrointestinal tract ( B )

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

PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the rumen, ileum, and colon, shown for the different segments of the gastrointestinal tract (B)


C . PCoA , on weighted UniFrac matrix , of 16S rRNA gene data or rumen , ileum , colon , combined data ( C ) . D . Microbial dissimilarity ( calculated on weighted UniFrac matrix , 100 values in each group ) between CtM groups in different intestinal compartments . lines , boxes , whiskers in box plot diagrams represent median , first third quartiles , min - to - max distribution of replicate values , respectively .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data or the rumen, ileum, and colon, combined data (C). D. Microbial dissimilarity (calculated based on the weighted UniFrac distance matrix, 100 values in each group) between the male and CtM groups in the different intestinal compartments. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively.


E . relative abundances of abundant taxa ( > 0 . 5 % of mean abundance ) .

E. The relative abundances of abundant bacterial taxa (> 0.5% of the mean abundance).


B . intestinal branched - chain acid ( BCAA ) in adult CtM Hanwoo ( n = 10 in each group ) . BCAA in luminal contents of ileum , cecum , colon were quantified by an enzymatic method using a BCAA kit . lines , boxes , whiskers in box plot diagrams represent median , first third quartiles , min - to - max distribution of replicate values , respectively . scattered dots in box plot diagrams represent replicates .

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

B. The intestinal branched-chain amino acid (BCAA) levels in the adult male and CtM Hanwoo (n = 10 in each group). BCAA levels in the luminal contents of the ileum, cecum, and colon were quantified by an enzymatic method using a BCAA assay kit. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates.


A . Representative of dressed bodies of adult CtM Hanwoo ( n = 10 in each group ) .

A. Representative images of dressed bodies of the adult male and CtM Hanwoo (n = 10 in each group).


B . Dressed , thickness of dorsal subcutaneous fat , total of extramuscular fat ( posterior subcutaneous fat , mesenteric fat , retroperitoneal fat ) , serum of branched - chain ( BCAAs ) .

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

B. Dressed body weight, thickness of the dorsal subcutaneous fat, total weight of the extramuscular fat (posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat), and serum levels of the branched-chain amino acids (BCAAs).


C . Representative of fresh striploin muscle from adult CtM carcasses ( n = 5 in each group ) .

C. Representative images of fresh striploin muscle from the adult male and CtM carcasses (n = 5 in each group).


D . intramuscular fat in - normalized muscle .

D. The intramuscular fat area in size-normalized muscle.


E . intramuscular metabolome profiles analyzed using GC - TOF - MS clustered by PCoA on Bray - Curtis dissimilarity

E. The intramuscular metabolome profiles analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix


F , G . relative abundances of intramuscular metabolites ( F ) β - hydroxybutyrate ( 3 - HB ) ( G ) in CtM samples .

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

F, G. The relative abundances of intramuscular metabolites (F) and β-hydroxybutyrate (3-HB) (G) in the male and CtM samples.


H . serum mucle ketone , quantified enzymatically , as described in Materials Methods section ( n = 10 in each group ) .

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

H. The serum and mucle ketone body levels, quantified enzymatically, as described in the Materials and Methods section (n = 10 in each group).


C . PCoA , on weighted UniFrac matrix , of 16S rRNA gene data for luminal contents of ileum colon , shown according to different diets antibiotic treatment .

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

C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon, shown according to the different diets and antibiotic treatment.


D . relative abundances of family Peptostreptococcaceae are displayed as a box dot plot .

D. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


E . serum branched - chain acid ( BCAA ) quantified enzymatically , as described in Materials Methods section .

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

E. The serum branched-chain amino acid (BCAA) levels quantified enzymatically, as described in the Materials and Methods section.


F . total of extramuscular fat , including posterior subcutaneous fat , mesenteric fat , retroperitoneal fat .

F. The total weight of the extramuscular fat, including posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat.


G , H . Representative ( G ) ( H ) of hindlimb intermuscular fat . are from a longitudinal section of hind leg . fat data are presented as a of .

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

G, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat. The images are from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum hindlimb muscle ketone quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels quantified enzymatically.


B . serum testosterone in Sham - R castrated ( CtM ) - R mice .

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

B. The serum testosterone levels in the Sham-R and castrated male (CtM)-R mice.


C . PCoA , on weighted UniFrac matrix , of 16S rRNA gene data for luminal contents of ileum colon .

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

C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon.


E . relative abundances of family Peptostreptococcaceae are displayed as a box dot plot .

E. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


F . Weights of posterior subcutaneous fat , epididymal fat , mesenteric fat , retroperitoneal fat in Sham - R CtM - R mice .

O 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. Weights of the posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat in the Sham-R and CtM-R mice.


G , H . Representative ( G ) ( H ) of hindlimb intermuscular fat in Sham - R CtM - R mice . were obtained from a longitudinal section of hind leg . fat data are presented as a of .

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

G, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat in the Sham-R and CtM-R mice. Images were obtained from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum hindlimb muscle ketone , quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


B . gain in response to different of dietary BCAAs .

B. Body weight gain in response to the different levels of dietary BCAAs.


C. Representative images of gross anatomy.


D . total of extramuscular fat ( posterior subcutaneous fat , epididymal fat , mesenteric fat , retroperitoneal fat ) in mice fed high - fat diet ( HFD ) with or without BCAAs .

O O O O O O O O O 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

D. The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high-fat diet (HFD) with or without BCAAs.


E . serum BCAA in mice fed HFD with or without BCAAs .

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

E. The serum BCAA levels in the mice fed HFD with or without BCAAs.


F . PCoA , on weighted UniFrac matrix , of 16S rRNA gene data for luminal contents of ileum colon are shown . gain data are presented as a of initial . fat data are presented as a of .

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

F. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.


G . Serum hindlimb muscle ketone , quantified enzymatically .

G. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


( B , Sequential dissociation of EI24 BiP from IRE1 elicits a two - phase activation of IRE1 in response to ER stress . were treated with 4 μM tunicamycin ( TM ) for indicated times . interactions of EI24 BiP with IRE1 were analyzed by

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

(B, Sequential dissociation of EI24 and BiP from IRE1 elicits a two-phase activation of IRE1 in response to ER stress. Cells were treated with 4 μM tunicamycin (TM) for the indicated times. The interactions of EI24 and BiP with IRE1 were analyzed by immunoprecipitation


( D ) EI24 knockout promotes activation of IRE1 , but not PERK or ATF6 . Control ( sgGFP ) EI24 knockout ( sgEI24 ) HeLa were treated with 4 μM TM for indicated times . expression was analyzed by immunoblotting .

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

(D) EI24 knockout promotes the activation of IRE1, but not PERK or ATF6. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were treated with 4 μM TM for the indicated times. Protein expression was analyzed by immunoblotting.


( G ) Expression of Flag - EI24 in EI24 knockout inhibits IRE1 activity . Flag - Metap2 served as control . transfected with indicated cDNAs were treated with or without 4 μM TM for 2 hr . expression was analyzed by immunoblotting .

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(G) Expression of Flag-EI24 in EI24 knockout cells inhibits IRE1 activity. Flag-Metap2 served as the control. Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr. Protein expression was analyzed by immunoblotting.


( B ) EI24 persistently localizes on ER regardless of presence or absence of ER stress . HeLa stably expressing Flag - EI24 were treated with or without 4 μM tunicamycin ( TM ) for 2 hr . Colocalization of EI24 with ER was analyzed . KDEL - mCherry , ER marker . Scale bar , 10 μm .

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(B) EI24 persistently localizes on the ER regardless of the presence or absence of ER stress. HeLa cells stably expressing Flag-EI24 were treated with or without 4 μM tunicamycin (TM) for 2 hr. Colocalization of EI24 with ER was analyzed. KDEL-mCherry, ER marker. Scale bar, 10 μm.


( D ) C - terminus of EI24 is required for IRE1 binding . HeLa transfected with indicated EI24 mutants were treated as in ( B ) , after which - interactions were analyzed by immunoprecipitation ( IP ) . star represents IgG chains . ( E ) C - terminus of EI24 binds to IRE1 in a persistent fashion . HeLa transfected with indicated EI24 domains were treated as in ( B ) . - interactions were analyzed by IP . star represents IgG chains . (

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

(D) The C-terminus of EI24 is required for IRE1 binding. HeLa cells transfected with the indicated EI24 mutants were treated as in (B), after which protein-protein interactions were analyzed by immunoprecipitation (IP). The star represents IgG light chains. (E) The C-terminus of EI24 binds to IRE1 in a persistent fashion. HeLa cells transfected with the indicated EI24 domains were treated as in (B). Protein-protein interactions were analyzed by IP. The star represents IgG light chains. (