HAPI cells were treated with RK - 33 ( 10 μM ) , HA14 - 1 ( 25 μM ) , or DMSO vehicle , followed by oligomycin ( oligo , 0 . 5 μg / ml ) , FCCP ( 3 μM ) , and antimycin A ( AA , 1 μM ) while oxygen consumption rate ( OCR ) was measured . Pyruvate ( 10 mM ) was added in combination with FCCP to ensure that substrate supply was not rate - limiting for maximal OCR . Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages . OCR is baseline - normalized to the point prior to drug or vehicle addition .

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HAPI cells were treated with RK-33 (10 μM), HA14-1 (25 μM), or DMSO vehicle, followed by oligomycin (oligo, 0.5 μg/ml), FCCP (3 μM), and antimycin A (AA, 1 μM) while oxygen consumption rate (OCR) was measured. Pyruvate (10 mM) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal OCR. Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages. OCR is baseline-normalized to the point prior to drug or vehicle addition.


HAPI microglial cells were incubated for 1 h in glucose - free XF24 assay medium that was supplemented with 2 - deoxyglucose ( 50 mM ) and pyruvate ( 10 mM ) . RK - 33 ( 10 μM ) , oligomycin ( 0 . 5 μg / ml ) , or vehicle control was additionally present as indicated . Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages . Significance was assessed by two - sided , unpaired t - test .

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HAPI microglial cells were incubated for 1 h in glucose-free XF24 assay medium that was supplemented with 2-deoxyglucose (50 mM) and pyruvate (10 mM). RK-33 (10 μM), oligomycin (0.5 μg/ml), or vehicle control was additionally present as indicated. Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages. Significance was assessed by two-sided, unpaired t-test.


Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1 / KrasG12D mouse . Scale bar is 100 μm .

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Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1/KrasG12D mouse. Scale bar is 100 μm.


Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( C ) , before treatment and 1 week after treatment . Tumors are indicated by arrows and confirmed by H & amp ; amp ; E staining of lung sections ( lower panel ) . Scale bar is 250 μm . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( D ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (C), before treatment and 1 week after treatment. Tumors are indicated by arrows and confirmed by H&amp;E staining of lung sections (lower panel). Scale bar is 250 μm.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (D). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in ( C ) . Figure displays H & amp ; amp ; E staining of lung sections from radiation - treated ( upper panel ) and RK - 33 - and radiation - treated mice ( lower panel ) . Scale bar is 2 mm . Quantification of tumor burden ( as tumor surface divided by total lung surface ) in orthotopic A549 lung cancer mouse model , as shown in ( F ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in (C). Figure displays H&amp;E staining of lung sections from radiation-treated (upper panel) and RK-33- and radiation-treated mice (lower panel). Scale bar is 2 mm.Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic A549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( H ) , before treatment and 1 week after treatment . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( I ) and expressed as relative tumor size . Significance was assessed by two - sided , unpaired t - test . Error bars represent SEM .

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Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (H), before treatment and 1 week after treatment.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (I) and expressed as relative tumor size. Significance was assessed by two-sided, unpaired t-test. Error bars represent SEM.


A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry . Knockdown of DDX3 led to a decrease of cells in S - phase and an increase of cells in G1 - phase , indicative of a G1 arrest . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry. Knockdown of DDX3 led to a decrease of cells in S-phase and an increase of cells in G1-phase, indicative of a G1 arrest. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


B , C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK - 33 ( 0 , 2 , 4 , and 6 μM ) . RK - 33 induced a G1 cell cycle arrest in both cell lines . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD .

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B, C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK-33 (0, 2, 4, and 6 μM). RK-33 induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.


D , E Immunoblot of cell cycle - related proteins ( Cyclin D1 and Cyclin E1 ) and cell death - related proteins ( cleaved caspase 7 , cleaved caspase 9 ) in A549 and H1299 cells after treatment with RK - 33 ( 10 μM ) . Initially , a strong decrease of Cyclin D1 was observed . After 8 and 24 h , cleaved caspases 9 and 7 were apparent .

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D, E Immunoblot of cell cycle-related proteins (Cyclin D1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in A549 and H1299 cells after treatment with RK-33 (10 μM). Initially, a strong decrease of Cyclin D1 was observed. After 8 and 24 h, cleaved caspases 9 and 7 were apparent.


F Immunoblot of MAPK pathway - related proteins ERK1 / 2 and phosphorylated ERK1 / 2 in A549 , H1299 , and H3255 ( RK - 33 resistant ) cells 24 h after treatment with RK - 33 ( 7 . 5 μM or 10 μM ) . ERK2 and especially ERK1 become dephosphorylated after treatment with RK - 33 in A549 and H1299 cells but not in H3255 cells . Outlined boxes indicate spliced lanes .

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F Immunoblot of MAPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in A549, H1299, and H3255 (RK-33 resistant) cells 24 h after treatment with RK-33 (7.5 μM or 10 μM). ERK2 and especially ERK1 become dephosphorylated after treatment with RK-33 in A549 and H1299 cells but not in H3255 cells. Outlined boxes indicate spliced lanes.


A β - catenin ( red ) and DDX3 ( green ) expression in H1299 cells . After overexpressing β - catenin , both DDX3 and β - catenin accumulate in the nucleus . Scale bar is 10 μm .

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A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.


B Immunoprecipitation with DDX3 or H3K4Me3 ( control ) and immunoblotted with β - catenin in A549 and H1299 cells . Outlined boxes indicate spliced lanes .

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B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.


C Immunoprecipitation with β - catenin or H3K4Me3 ( control ) and immunoblotted with DDX3 in A549 and H1299 cells . Outlined boxes indicate spliced lanes .

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C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.


D , E β - catenin / TCF4 activity was determined by the TOP / FOP reporter assay . Co - transfection with β - catenin is indicated below .

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D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.


F - I H1299 and A549 cells were treated with RK - 33 ( 0 , 1 , 2 , and 3 μM ) and co - transfected with β - catenin in ( F , H ) . Treatment with RK - 33 decreased TCF4 activity in both cell lines .

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F-I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.


J , K Normalized mRNA expression of TCF4 - regulated proteins ( Axin - 2 , c - Myc , Cyclin D1 ) and DDX3 were measured by qRT - PCR in H1299 cells after knockdown of DDX3 ( J ) and treatment with RK - 33 ( K ) . All experiments were repeated three times .

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J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT-PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.


A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2 - Gy radiation and A549 cells pre - treated with 6 μM RK - 33 , 12 h before radiation . Overlap of 53BP1 and γH2AX is seen in the merged picture of the co - immunofluorescence staining . Scale bar is 2 μm .

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A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.


B A549 cells were pre - treated with RK - 33 and radiated with 2 Gy , and 53BP1 and γH2AX foci were counted as a measure of DNA damage . Cells with more than 10 foci 53BP1 or γH2AX were counted . More than 400 cells per sample were evaluated .

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B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.


C H1299 cells stably transfected with a homologous recombination ( HR ) reporter construct were treated with RK - 33 . Reporter constructs expressed GFP , which was quantified by flow cytometry . Experiments were repeated three times .

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C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.


D H1299 cells , containing a stable non - homologous end - joining ( NHEJ ) reporter construct , were treated with RK - 33 and knockdown of DDX3 . Reporter construct expressed GFP , which was quantified by flow cytometry . All experiments were repeated three times .

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D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.


E Microarray results from MDA - MB - 231 cells treated with RK - 33 and shDDX3 were validated by qRT - PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates ( Bio - Rad ) and performed in biological triplicates .

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E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT-PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.


F , G DNA repair - related proteins ( ATR and XRCC4 ) , DDX3 , and actin were assessed by immunoblotting in A549 ( F ) and H1299 ( G ) cells . Cells were pretreated for 4 h with vehicle control or 6 μM RK - 33 and then radiated with 0 or 5 Gy . Outlined boxes indicate spliced lanes .

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F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.


ATPase staining of GAS and SOL muscles of WT and DKO mice ( left ) . Corresponding percentage of type I fibers were determined ( right ) ( n = 5 ) . Scale bars : 100 μm .

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ATPase staining of GAS and SOL muscles of WT and DKO mice (left). Corresponding percentage of type I fibers were determined (right) (n=5). Scale bars: 100 μm.


Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice ( n = 3 for PGC1α analysis ; n = 5 for PLIN5 analysis ) .

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Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice (n=3 for PGC1α analysis; n=5 for PLIN5 analysis).


Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR - 183 and miR - 96 agomirs ( Ago - 183 / 96 ) or antagomirs ( Ant - 183 / 96 ) ( n = 3 ) .

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Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR-183 and miR-96 agomirs (Ago-183/96) or antagomirs (Ant-183/96) (n=3).


Mitochondrial respiration profile of C2C12 cells transfected with Ago - 183 / 96 ( J ) or Ant - 183 / 96 ( K ) . Cellular oxygen consumption rate ( OCR ) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR ( n = 3 or 4 per group ) .

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Mitochondrial respiration profile of C2C12 cells transfected with Ago-183/96 (J) or Ant-183/96 (K). Cellular oxygen consumption rate (OCR) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR (n=3 or 4 per group).


Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in GAS muscles of WT and DKO mice . Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) .

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Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in GAS muscles of WT and DKO mice. Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).


Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice . Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) .

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Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice. Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).


LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR - 183 / 96 ( N ) or antagomir - 183 / 96 ( P ) stained with Nile red ( n = 5 ) . Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR - 183 / 96 ( O ) or antagomir - 183 / 96 ( Q ) . Scale bars : 5 μm .

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LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR-183/96 (N) or antagomir-183/96 (P) stained with Nile red (n=5). Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR-183/96 (O) or antagomir-183/96 (Q). Scale bars: 5 μm.


Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 agomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 . Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 antagomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 agomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 antagomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96.


Relative mRNA expression ( I , left ) and western blot analysis ( J , top ) of ATGL in C2C12 cells transfected with agomir for miR - 183 ( Ago - 183 ) or miR - 96 ( Ago - 96 ) . Relative mRNA expression ( I , right ) and western blot analysis ( J , bottom ) of ATGL in C2C12 cells transfected with antagomir for miR - 183 ( Ant - 183 ) or miR - 96 ( Ant - 96 ) . ( n = 5 per group for RT - PCR analysis )

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O

Relative mRNA expression (I, left) and western blot analysis (J, top) of ATGL in C2C12 cells transfected with agomir for miR-183 (Ago-183) or miR-96 (Ago-96). Relative mRNA expression (I, right) and western blot analysis (J, bottom) of ATGL in C2C12 cells transfected with antagomir for miR-183 (Ant-183) or miR-96 (Ant-96). (n=5 per group for RT-PCR analysis)


Relative mRNA expression of ATGL , PGC1α , PLIN5 , and HSL in C2C12 cells transfected with ATGL ( n = 5 ) .

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Relative mRNA expression of ATGL, PGC1α, PLIN5, and HSL in C2C12 cells transfected with ATGL (n=5).


Relative mRNA expression of PGC1α , PLIN5 , and HSL in C2C12 cells transfected with Ant - 183 / 96 and siRNA of PPARδ ( siPPARδ ) .

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Relative mRNA expression of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siRNA of PPARδ (siPPARδ).


Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice ( n = 3 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice .

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Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice (n=3). Western blot analysis of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice.


Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months ( n = 5 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O O

Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months (n=5). Western blot analysis of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months.


Concentration of serum triglyceride ( TAG ) ( left ) and non - esterified fatty acid ( NEFA ) ( right ) in WT and DKO mice after HFD feeding for 2 months ( n = 5 ) .

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Concentration of serum triglyceride (TAG) (left) and non-esterified fatty acid (NEFA) (right) in WT and DKO mice after HFD feeding for 2 months (n=5).


Western blot analysis of FoxO1 , PDK4 , p - PDHA1 , PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months .

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Western blot analysis of FoxO1, PDK4, p-PDHA1, PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months.


Glucose tolerance test ( GTT ) and area under the curve ( AUC ) data for GTT in WT and DKO mice after HFD feeding ( n = 10 - 11 ) .

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Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).


( B ) Correlation between replicate mock infections . Path - seq reads were recovered from samples of macrophage RNA spiked with 0 . 1 % MTB RNA . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 . Inset summarizes the mock infection replicates and their fraction of MTB reads ( of all aligned reads ) from Path - seq and standard RNA - seq methods .

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(B) Correlation between replicate mock infections. Path-seq reads were recovered from samples of macrophage RNA spiked with 0.1% MTB RNA. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001. Inset summarizes the mock infection replicates and their fraction of MTB reads (of all aligned reads) from Path-seq and standard RNA-seq methods.


( C ) Correlation between MTB RNA sequenced by RNA - seq ( Illumina TruSeq library prep ) and the same MTB RNA sample combined with macrophage RNA at 1 : 1000 ratio and processed using Path - seq method . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 .

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(C) Correlation between MTB RNA sequenced by RNA-seq (Illumina TruSeq library prep) and the same MTB RNA sample combined with macrophage RNA at 1:1000 ratio and processed using Path-seq method. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001.


( A ) Path - seq profiles of desA1 , desA2 in MTB infected BMDMs . Error bars show the standard deviation from three biological samples . Representative results from two experiments are presented .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-ORGANISM B-DISEASE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

(A) Path-seq profiles of desA1, desA2 in MTB infected BMDMs. Error bars show the standard deviation from three biological samples. Representative results from two experiments are presented.


( B ) Upper panel : dissolved oxygen curve over 120 h time course showing controlled depletion ( 1 ) , sustained hypoxia ( 2 ) and controlled reaeration ( 3 ) . Points represent the average of three biological replicates and were measured via fiber optic technology that non - invasively probes oxygen levels in the culture ( PreSens Precision Sensing GmbH ) . Lower panel : expression profiles ( RNA - seq ) of desA1 and desA2 over the time course and oxygen levels . Error bars show the standard deviation from three biological samples .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-DISEASE O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(B) Upper panel: dissolved oxygen curve over 120 h time course showing controlled depletion (1), sustained hypoxia (2) and controlled reaeration (3). Points represent the average of three biological replicates and were measured via fiber optic technology that non-invasively probes oxygen levels in the culture (PreSens Precision Sensing GmbH). Lower panel: expression profiles (RNA-seq) of desA1 and desA2 over the time course and oxygen levels. Error bars show the standard deviation from three biological samples.


( B ) Heatmap of TFs with decreased ( purple ) or increased ( green ) activity at specific time points during in vitro or in vivo infection .

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(B) Heatmap of TFs with decreased (purple) or increased (green) activity at specific time points during in vitro or in vivo infection.


( C ) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity ( induced expression of target genes ) . ( D ) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity ( de - represses target genes ) . ( E ) Rv0691c regulon genes differentially expressed in vitro and in vivo ; evidence for increased Rv0691c activity ( increased up - and down - regulation of target genes ) at 24 h from both in vitro and in vivo infection .

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(C) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity (induced expression of target genes). (D) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity (de-represses target genes). (E) Rv0691c regulon genes differentially expressed in vitro and in vivo; evidence for increased Rv0691c activity (increased up- and down-regulation of target genes) at 24 h from both in vitro and in vivo infection.


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) and 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

(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 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 .

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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 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 .

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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 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 .

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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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O O B-EXP_ASSAY O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O B-ORGANISM O O O

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 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 .

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O O O

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

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O B-GENEPROD O O O O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-DISEASE B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O B-DISEASE B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O 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, 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 , 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-DISEASE O B-DISEASE O O O O O O O O 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-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-DISEASE O B-DISEASE O O

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 ) 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O B-DISEASE B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O

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 , 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 .

O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O O O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O O O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O

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 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 .

O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 − 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 .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(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 − 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 .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O

(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 .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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 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 .

O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O 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) 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 analysis of ASC and 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 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 .

O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O 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) 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 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 .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

(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 ) .

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


( 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 .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) 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 .

O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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, 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 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 .

O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O

(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 line tracing analysis from images in ( D ) .

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


( 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 .

O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(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 − 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 .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O

(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 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 .

O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(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 .

(B) Line tracing analysis of fluorescence signal intensity.


( 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 .

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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 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 .

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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 analysis of Rab8a knockdown in BMMs .

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(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 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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O 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) 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 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 .

O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O

(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 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 .

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

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


( 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 .

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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 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 .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O 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, 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 , 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 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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, 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 − 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 .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(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 − 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 .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O

(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 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 .

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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 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 ) .

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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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

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 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 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O 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-SMALL_MOLECULE 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-GENEPROD O O O O O

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 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 ) .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O 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.