A . Representative FACS plots of freshly isolated epidermal ( Epi d0 ) day 14 ( d14 ) cultures of epidermal in different growth conditions media . KGM 2D : 2D in basal KGM ; 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . B . CD34 + α6 + were quantified by flow cytometry from d14 as in ( A ) ( mean ± SD ; n = 3 - 5 ; * p ≤ 0 . 05 ; * * * p ≤ 0 . 001 , Kruskal - Wallis / Dunn ' s post ) . 3C 2D : 2D in KGM + Y27632 + VEGF - A + FGF - 2 ; Y : 3D - Matrigel in KGM + Y27632 ; YV : Y + VEGF - A ; YF : Y + FGF - 2 ; 3C : Y + VEGF - A + FGF - 2 . C . Absolute numbers of CD34 + α6 + from Epi d0 d14 cultures as in ( A - B ) . Fold enrichment over Epi d0 is shown ( mean ± , n = 3 - 5 ; * p ≤ 0 . 05 , Mann - Whitney U ) .

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A. Representative FACS plots of freshly isolated epidermal cells (Epi d0) and day 14 (d14) cultures of epidermal cells in different growth conditions and media. KGM 2D: 2D in basal KGM; 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. CD34+α6+ cells were quantified by flow cytometry from d14 as in (A) (mean ±SD; n=3-5; *p ≤ 0.05; ***p ≤ 0.001, Kruskal-Wallis/Dunn's post test). 3C 2D: 2D in KGM + Y27632 + VEGF-A + FGF-2; Y: 3D-Matrigel in KGM + Y27632; YV: Y + VEGF-A; YF: Y + FGF-2; 3C: Y + VEGF-A + FGF-2. C. Absolute numbers of CD34+α6+ cells from Epi d0 and d14 cultures as in (A-B). Fold enrichment over Epi d0 is shown (mean ±SEM, n=3-5; *p ≤ 0.05, Mann-Whitney U test).


D . Epidermal grown for 14 days in indicated conditions . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . 3C 2D : 2D in 3C medium . Scale bars 30µm .

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D. Epidermal cells grown for 14 days in the indicated conditions. 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. 3C 2D: 2D in 3C medium. Scale bars 30µm.


E . Immunofluorescence of 3C cultures showing expression of HFSC markers CD34 SOX9 in a subset of . See also Appendix Appendix figure S1 .

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E. Immunofluorescence analysis of 3C cultures showing the expression of the HFSC markers CD34 and SOX9 in a subset of cells. See also Appendix Appendix figure S1.


A . Colony forming assays show increased proliferative potential of cultured in 3C compared to freshly isolated epidermal ( Epi d0 ; control ) ( mean ± ; n = 5 ; * p ≤ 0 . 05 , Mann - Whitney U - ) . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 .

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A. Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells (Epi d0; control) (mean ±SEM; n=5; *p ≤ 0.05, Mann-Whitney U-test). 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2.


B . Full thickness skin reconstitution with freshly isolated epidermal ( Epi d0 ) or 3C cultures shows that cultured in 3C retain their self - renewal capacity multipotency . A representative recipient of 4 mice / condition is shown . Right panel shows of hair follicle density . Note that 3C cultures give rise to more hair compared to controls ( mean ± ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U ) .

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B. Full thickness skin reconstitution assay with freshly isolated epidermal cells (Epi d0) or 3C cultures shows that cells cultured in 3C retain their self-renewal capacity and multipotency. A representative recipient of 4 mice/condition is shown. Right panel shows quantification of hair follicle density. Note that 3C cultures give rise to more hair compared to controls (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).


C . Long - term culture of in 3C was performed as shown in left panel . 3C cultures maintain a stable population of CD34 + α6 + long term ( data are shown using a Box - - Whisker plot : box indicates 25th 75th percentiles ; error bars represent 10th 90th percentiles ; middle is median ; n = 3 - 5 independent experiments ) . p : passage .

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C. Long-term culture of cells in 3C was performed as shown in the left panel. 3C cultures maintain a stable population of CD34+α6+ long term (data are shown using a Box-and-Whisker plot: box indicates 25th and 75th percentiles; error bars represent 10th and 90th percentiles; middle line is the median; n=3-5 independent experiments). p: passage.


D . Full thickness skin reconstitution using 3C from passage 1 ( p1 ) passage 15 ( p15 ) show that cultured in 3C maintain their multipotency self - renewal capacity in long - term cultures . A representative recipient of 4 mice / condition is shown . See also Appendix figure S2 .

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D. Full thickness skin reconstitution assay using 3C cells from passage 1 (p1) and passage 15 (p15) show that cells cultured in 3C maintain their multipotency and self-renewal capacity in long-term cultures. A representative recipient of 4 mice/condition is shown. See also Appendix figure S2.


B . Euclidian dendrogram of quantified transcripts from RNAseq data generated as shown in panel A . 3C cultures cluster with purified CD34 + α6 + HFSCs ( n = 3 biological replicates ) .

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B. Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A. 3C cultures cluster with purified CD34+α6+ HFSCs (n=3 biological replicates).


E . of selected genes differentially expressed in 3C - CD34 + α6 + 3C - CD34 ¯ α6 + . Note upregulation of multiple key HFSC lineage identity transcription factors bulge markers ( triangles ) in 3C - CD34 + α6 + , upregulation of genes implicated in lineage progression in 3C - CD34 ¯ α6 + ( circles ) .

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E. Heatmap of selected genes differentially expressed in 3C-CD34+α6+ and 3C-CD34¯α6+ cells. Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers (triangles) in 3C-CD34+α6+ cells, and upregulation of genes implicated in lineage progression in 3C-CD34¯α6+ cells (circles).


G . RT - qPCR analyses of FACS - purified CD34 + α6 + CD34 - α6 + from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated 3C - CD34 + α6 + compared to freshly isolated CD34 - α6 + progenitors . 3C - CD34 - α6 + show intermediated expression ( mean ± ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) .

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G. RT-qPCR analyses of FACS-purified CD34+α6+ and CD34-α6+ cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C-CD34+α6+ cells compared to freshly isolated CD34-α6+ progenitors. 3C- CD34-α6+ cells show intermediated expression (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).


H . Both 3C - CD34 + α6 + 3C - CD34 - α6 + show upregulation of a panel of cycle genes genes shown to be enriched in HFSC progeny ( mean ± ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S3 Tables S1 - S3 .

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H. Both 3C-CD34+α6+ and 3C-CD34-α6+ cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S3 and Tables S1-S3.


A . FACS of CD34 + α6 + over time shows that 3C cultures reach a ~ 50 : 50 population equilibrium after 10 days of culture ( mean ± ; n = 3 ) .

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A. FACS analysis of CD34+α6+ cells over time shows that 3C cultures reach a ~50:50 population equilibrium after 10 days of culture (mean ±SEM; n=3).


B . were EdU - labeled for 24 h ( day 10 - 11 ) prior to by flow cytometry to quantify proliferating . of EdU + is indicated ( n = 4 ; mean ± SD ; * p ≤ 0 . 05 , Mann - Whitney U - ) .

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B. Cells were EdU-labeled for 24 h (day 10-11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test).


D . Simulated population proportions of CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs derived from mathematical model describing steady state cultures . Experimental data from ( A ) are shown as reference ( in grey ) . See also Appendix figure S4 Dataset EV4 .

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D. Simulated population proportions of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs derived from the mathematical model describing steady state cultures. Experimental data from (A) are shown as reference (in grey). See also Appendix figure S4 and Dataset EV4.


A . CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs were FACS - purified from total epidermis ( d0 ) to ≥ 98 % purity post sorting ( d0 post - sort ) , grown 14 days in 3C cultures ( d14 3C ) . A significant population of CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs is generated from both purified populations after 14 days . A representative of 3 independent experiments is shown ( mean ± SD ) .

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A. CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs were FACS-purified from total epidermis (d0) to ≥98% purity post sorting (d0 post-sort), and grown 14 days in 3C cultures (d14 3C). A significant population of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs is generated from both purified populations after 14 days. A representative of 3 independent experiments is shown (mean ±SD).


B - C . Time course analyses of 3C cultures established from FACS - purified populations of CD34 + α6 + HFSCs ( B ) or CD34 ¯ α6 + non - HFSCs ( C ) from 3C cultures . Both cultures reach population equilibrium between day 6 10 ( n = 4 ; mean ± SD ) .

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B-C. Time course analyses of 3C cultures established from FACS-purified populations of CD34+α6+ HFSCs (B) or CD34¯α6+ non-HFSCs (C) from 3C cultures. Both cultures reach population equilibrium between day 6 and 10 (n=4; mean ±SD).


D - E . Simulated population proportions of CD34 + α6 + HFSCs ( D ) CD34 ¯ α6 + non - HFSCs ( E ) from mathematical model describing behavior of pure populations . model parameters were estimated from experimental data in B - C ( shown in grey as reference ) .

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D-E. Simulated population proportions of CD34+α6+ HFSCs (D) and CD34¯α6+ non-HFSCs (E) from the mathematical model describing behavior of pure cell populations. The model parameters were estimated from the experimental data in B-C (shown in grey as reference).


G . Lineage tracing of FACS - purified CD34 + α6 + HFSCs CD34 ¯ α6 + non - HFSCs from EGFP ¯ EGFP + 3C cultures , respectively . CD34 + α6 + CD34 ¯ α6 + were mixed at indicated ratios at d0 EGFP expression was used to trace of origin over time by flow cytometry . Presence of CD34 + α6 + EGFP + in cultures at d10 indicates non - HFSC to HFSC interconversion .

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G. Lineage tracing of FACS-purified CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs from EGFP¯ and EGFP+ 3C cultures, respectively. CD34+α6+ and CD34¯α6+ were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry. Presence of CD34+α6+ EGFP+ cells in cultures at d10 indicates non-HFSC to HFSC interconversion.


H . Lineage tracing as in ( F ) shows that 3C cultures reach equilibrium between day 6 10 independent of initial proportions of HFSCs non - HFSCs .

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H. Lineage tracing as in (F) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non-HFSCs.


I . of proportion of CD34 + α6 + that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled are indicated ) .

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I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated).


J . of proportion of CD34 ¯ α6 + that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled are indicated ) . Data in panels G - J show n = 4 ; mean ± SD . See also Appendix figure S5 .

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J. Quantification of the proportion of CD34¯α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). Data in panels G-J show n=4; mean ±SD. See also Appendix figure S5.


A - B . Inhibitor treatment scheme ( A ) FACS plots ( B ) of 3C cultures treated with Shh inhibitor cyclopamine or BMP inhibitor dorsomorphin ( mean ± SD ; n = 3 ) . C . of experiments as in ( B ) shows enrichment of HFSCs upon cyclopamine ( Cyclo ) treatment , whereas dorsomorphin ( Dorso ) treatment leads to depletion of HFSCs ( mean ± SD ; n = 3 ; * p ≤ 0 . 05 , Kruskal - Wallis ) .

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A-B. Inhibitor treatment scheme (A) and FACS plots (B) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin (mean ±SD; n=3). C. Quantification of experiments as in (B) shows enrichment of HFSCs upon cyclopamine (Cyclo) treatment, whereas dorsomorphin (Dorso) treatment leads to depletion of HFSCs (mean ±SD; n=3; *p ≤ 0.05, Kruskal-Wallis).


D . of significantly ( padj value < 0 . 05 ) differentially expressed genes from RNAseq of 3C cultures treated with Cyclo or Dorso for 48h . HFSC identity genes ( triangles ) genes implicated in HFSC lineage progression ( circles ) are indicated .

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D. Heatmap of significantly (padj value<0.05) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h. HFSC identity genes (triangles) and genes implicated in HFSC lineage progression (circles) are indicated.


E . RT - qPCR of 3C cultures treated as in ( D ) confirms that Id 1 , 2 3 are significantly upregulated in Cyclo treated ( mean ± ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U ) .

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E. RT-qPCR analysis of 3C cultures treated as in (D) confirms that Id 1,2 and 3 are significantly upregulated in Cyclo treated cells (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).


F . RT - qPCR of 3C cultures treated as in ( A ) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment ( mean ± ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) .

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F. RT-qPCR analysis of 3C cultures treated as in (A) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).


H . of experiments as in E ( mean ± SD ; n = 3 - 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S6 Dataset EV5 .

H. Quantification of experiments as in E (mean ±SD; n=3-4; * p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S6 and Dataset EV5.


A qPCR of relative SCTR in different tissues [ heart ( He ) , intestine ( Int ) , inguinal WAT ( Ing ) , epididymal WAT ( Ep ) , brown fat adipose ( BAT ) , liver ( Liv ) , hypothalamus ( Hypo ) , placenta ( Pla ) , skeletal muscle ( Mus ) , kidney ( Kid ) ] from C57BL / 6J mice as indicated ( n = 5 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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A qPCR analysis of relative SCTR levels in different tissues [heart (He), intestine (Int), inguinal WAT (Ing), epididymal WAT (Ep), brown fat adipose (BAT), liver (Liv), hypothalamus (Hypo), placenta (Pla), skeletal muscle (Mus), kidney (Kid)] from C57BL/6J mice as indicated (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


B Correlation of serum SCT serum triglycerides ( TG ) , total cholesterol ( TC ) nonesterified fatty ( NEFA ) in pregnant women in middle trimester to early third trimester ( n = 15 ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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B Correlation of serum SCT levels and serum triglycerides (TG), total cholesterol (TC) and nonesterified fatty acids (NEFA) levels in pregnant women in the middle trimester to the early third trimester (n = 15). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


C , D Plasma SCT of dams ( C ) at gestational day 18 . 5 ( GD18 . 5 ) ( D ) at E18 . 5 ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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C, D Plasma SCT levels of dams (C) at gestational day 18.5 (GD18.5) and embryo (D) at E18.5 (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


E Representative of SCT immunohistochemical of duodenum from dams on standard ( CON ) , whole grain ( WG ) high fat ( HFD ) diet at GD18 . 5 . SCT expression of WG is highest , but lower in HFD compared with CON . Scale bar , 50 μm ( n = 10 per group ) . Right part shows of SCT positive in duodenum from dams of three groups . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 .

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E Representative images of SCT immunohistochemical staining of duodenum from dams on standard (CON), whole grain (WG) and high fat (HFD) diet at GD18.5. SCT expression of WG is highest, but lower in HFD compared with CON. Scale bar, 50 μm (n = 10 per group). Right part shows quantification of SCT positive cells in duodenum from dams of three groups. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.


B Change of of M - SCT floxed M - SCT KO mice upon HFD feeding ( n = 8 per group ) . Right part shows representative photograph of M - SCT floxed M - SCT KO mice with HFD . Data information : Statistical significance was determined using Two - Student ' s t .

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B Change of body weight of M-SCT floxed and M-SCT KO mice upon HFD feeding (n = 8 per group). Right part shows representative photograph of M-SCT floxed and M-SCT KO mice with HFD. Data information: Statistical significance was determined using Two-tailed Student's t test.


C of different tissues from M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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C The weight of different tissues from M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


D Random blood glucose in M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . E Insulin in M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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D Random blood glucose levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). E Insulin levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


F Intraperitoneal glucose tolerance ( GTT ) ( 1 . 0 g glucose / kg ) in M - SCT floxed M - SCT KO mice with HFD ( n = 5 per group ) AUCs were calculated . Mice were fasted for 18h before . G Intraperitoneal insulin tolerance ( ITT ) ( 0 . 75 U insulin / kg ) in M - SCT floxed M - SCT KO mice with HFD ( n = 5 per group ) AUCs were calculated . mice were fasted for 6h before assays . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Intraperitoneal glucose tolerance test (GTT) (1.0 g glucose/ kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. Mice were fasted for 18h before assay. G Intraperitoneal insulin tolerance test (ITT) (0.75 U insulin/kg body weight) in M-SCT floxed and M-SCT KO mice with HFD (n = 5 per group) and AUCs were calculated. mice were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


H Serum TG , TC NEFA in M - SCT floxed M - SCT KO mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Serum TG, TC and NEFA levels in M-SCT floxed and M-SCT KO mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.


A , B Time - resolved oxygen consumption ( A ) heat production ( B ) in 48 - hour / cycle measured by CLAMS in M - SCT floxed M - SCT KO mice with HFD ( n = 6 per group ) , phase is marked as background . Regression - absolute oxygen consumption ( A ) or heat production ( B ) of are shown in right part . Data information : Statistical significance was determined using Two - Student ' s t . Data in panel A B have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Time-resolved oxygen consumption (A) and heat production (B) in 48-hour light/dark cycle measured by CLAMS in M-SCT floxed and M-SCT KO mice with HFD (n = 6 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (A) or heat production (B) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel A and B have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


C of 8 - week - old M - SCT floxed M - SCT KO offspring with chow diet during cold exposure ( 4 ℃ ; n = 8 ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Body temperature of 8-week-old M-SCT floxed and M-SCT KO male offspring with chow diet during cold exposure (4℃; n = 8). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


D Representative HE UCP1 of Ing from M - SCT floxed M - SCT KO mice with chow diet maintained at RT or 3 days of cold exposure ( 4 ℃ ) . Scale bar , 100 μm .

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

D Representative HE staining and UCP1 staining of Ing from M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or 3 days of cold exposure (4℃). Scale bar, 100 μm.


E Relative mRNA of UCP1 , CIDEA , PRDM16 , PGC1α DIO2 in iWAT from M - SCT floxed M - SCT KO mice with chow diet ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative mRNA levels of UCP1, CIDEA, PRDM16, PGC1α and DIO2 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


F Representative western blots of UCP1 , PRDM16 , PGC1α HSP90 in iWAT from M - SCT floxed M - SCT KO mice with chow diet after 3 days of cold exposure ( 4 ℃ ) . G Total UCP1 per mg iWAT of M - SCT floxed M - SCT KO mice with chow diet maintained at RT or exposed to cold for 3 d ( 4 ℃ ) ( n = 3 ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative western blots of UCP1, PRDM16, PGC1α and HSP90 in iWAT from M-SCT floxed and M-SCT KO mice with chow diet after 3 days of cold exposure (4℃). G Total UCP1 protein per mg iWAT of M-SCT floxed and M-SCT KO mice with chow diet maintained at RT or exposed to cold for 3 d (4℃) (n = 3). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


H Relative mRNA of UCP1 , PGC1α , PRDM16 , CIDEA DIO2 in SVF isolated from M - SCT KO M - SCT floxed mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in SVF isolated from M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


I , J OCR of Oligomycin , FCCP Antimycin A / Rotenone - treated matured adipocytes derived from iWAT - SVF of M - SCT KO M - SCT floxed mice with HFD , AUC of OCR as indicated were calculated ( n = 5 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

I, J OCR of Oligomycin, FCCP and Antimycin A/Rotenone-treated matured adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD, and the AUC of OCR as indicated were calculated (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


K Glucose oxidation in adipocytes derived from iWAT - SVF of M - SCT KO M - SCT floxed mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

K Glucose oxidation in adipocytes derived from iWAT-SVF of M-SCT KO and M-SCT floxed mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


B Stacked bars showing Proportional distribution of hypo - hypermethylated DMRs ( M - SCT KO vs M - SCT floxed ) in different regions . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Stacked bars showing the Proportional distribution of hypo- and hypermethylated DMRs (M-SCT KO vs M-SCT floxed) in different regions. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


C Heat map of DMR methylation level in mRNA between M - SCT floxed M - SCT KO . color bar indicates normalized tag in M - SCT floxed or M - SCT KO . Positive z scores , shown in table , indicate hypermethylated regions , while negative z scores imply hypomethylated regions . D As in ( C ) , for enhancer region .

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

C Heat map of DMR methylation level in mRNA between M-SCT floxed and M-SCT KO. The color bar indicates the normalized tag counts in M-SCT floxed or M-SCT KO. Positive z scores, shown in the table, indicate hypermethylated regions, while negative z scores imply hypomethylated regions. D As in (C), for enhancer region.


E Scatter plot representation of most significant difference of methylation in mRNA ( left ) enhancer ( right ) regions between M - SCT floxed M - SCT KO ( p < 0 . 005 ) . Data information : Statistical significance was determined using Two - Student ' s t .

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

E Scatter plot representation of the most significant difference of methylation in mRNA (left) and enhancer (right) regions between M-SCT floxed and M-SCT KO (p < 0.005). Data information: Statistical significance was determined using Two-tailed Student's t test.


F Volcano plot methylation represents DMRs in mRNA ( left ) enhancer ( right ) regions . Cutoffs ( vertical horizontal dashed lines ) used to identify DMRs between M - SCT floxed M - SCT KO mice . vertical lines correspond to twofold up down , respectively , horizontal represents a P value of 0 . 01 . Data information : Statistical significance was determined using Two - Student ' s t .

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

F Volcano plot DNA methylation represents all DMRs in mRNA (left) and enhancer (right) regions. Cutoffs (vertical and horizontal dashed lines) used to identify DMRs between M-SCT floxed and M-SCT KO mice. The vertical lines correspond to twofold up and down, respectively, and the horizontal line represents a P value of 0.01. Data information: Statistical significance was determined using Two-tailed Student's t test.


I , J Schematic diagram showing CpG islands of Mus musculus PRDM16 ( I ) CIDEA ( J ) genes . conditions to be met by CpG islands identified were : 200 - 300 bp , CG content of > 50 % observed CpG content vs expected CpG content > 0 . 6 . Methylation of each CpG islands in PRDM16 ( I ) CIDEA ( J ) are presented as fold change relative to average value of M - SCT floxed group β - actin promoter was used as a negative control ( n = 3 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not statistically significant .

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

I, J Schematic diagram showing the CpG islands of Mus musculus PRDM16 (I) and CIDEA (J) genes. The conditions to be met by CpG islands identified were: 200-300 bp length, CG content of > 50% and observed CpG content vs expected CpG content > 0.6. Methylation levels of each CpG islands in PRDM16 (I) and CIDEA (J) are presented as the fold change relative to the average value of the M-SCT floxed group and β-actin promoter was used as a negative control (n = 3 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.


A Western blots of DNMT1 , DNMT3a , DNMT3b HSP90 in Ing from M - SCT KO M - SCT floxed mice with HFD .

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

A Western blots analysis of DNMT1, DNMT3a, DNMT3b and HSP90 in Ing from M-SCT KO and M-SCT floxed mice with HFD.


B Relative mRNA of DNMT1 , DNMT3a DNMT3b in Ing from M - SCT KO M - SCT floxed mice with HFD ( n = 8 ) . Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . ns , not statistically significant .

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

B Relative mRNA levels of DNMT1, DNMT3a and DNMT3b in Ing from M-SCT KO and M-SCT floxed mice with HFD (n = 8). Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. ns, not statistically significant.


C Western blots of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in of M - SCT KO M - SCT floxed mice at E18 . 5 .

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

C Western blots analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in embryo of M-SCT KO and M-SCT floxed mice at E18.5.


D Western blot of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in mouse fibroblast ( MEF ) treated with 10 nM or 100 nM SCT .

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

D Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in mouse embryo fibroblast (MEF) cells treated with 10 nM or 100 nM SCT.


E Western blot of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in MEF treated with 10 nM SCT or 20 μM compound C ( C . C . ) for 24 h .

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

E Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in MEF cells treated with 10 nM SCT or 20 μM compound C (C.C.) for 24 h.


F Western blot of p - AMPKα , AMPKα , DNMT1 , SCTR HSP90 . MEF were transfected with indicated siRNA then treated with 1 mM AMPK agonist ( AICAR ) for 24 h .

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

F Western blot analysis of p-AMPKα, AMPKα, DNMT1, SCTR and HSP90. MEF cells were transfected with indicated siRNA and then treated with 1 mM AMPK agonist (AICAR) for 24 h.


G Western blot of p - AMPKα , AMPKα , p - ACC , ACC , DNMT1 HSP90 in AMPK + / + or AMPKα1 / α2 double knockout ( AMPK - / - ) MEF treated with 10 nM SCT for 24 h .

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

G Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1 and HSP90 in AMPK+/+ or AMPKα1/α2 double knockout (AMPK-/-) MEF cells treated with 10 nM SCT for 24 h.


H , I Western blot of Flag - DNMT1 in Flag - DNMT1 - transfected HEK293T treated with AICAR with indicated dose or time periods .

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

H, I Western blot analysis of Flag-DNMT1 in Flag-DNMT1-transfected HEK293T cells treated with AICAR with indicated dose or time periods.


J HEK293T were transfected with indicated plasmids then treated with 10 nM SCT for 24 h , followed by IP IB .

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

J HEK293T cells were transfected with indicated plasmids and then treated with 10 nM SCT for 24 h, followed by IP and IB analysis.


K HEK293T were transfected with indicated plasmids or siRNA then lysed , followed by IP IB .

K HEK293T cells were transfected with indicated plasmids or siRNA and then lysed, followed by IP and IB analysis.


L AMPK + / + or AMPK - / - MEF were transfected with indicated plasmids then lysed , followed by IP IB .

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

L AMPK+/+ or AMPK-/- MEF cells were transfected with indicated plasmids and then lysed, followed by IP and IB analysis.


A Serum SCT in M - HFD M - HFD + SCT mice with HFD ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Serum SCT levels in M-HFD and M-HFD+SCT mice with HFD (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


B Change of of M - HFD ( offspring of dams with high - fat pregnant diets ) M - HFD + SCT ( offspring of dams with high - fat pregnant diets supplied with SCT ) mice upon HFD feeding ( n = 8 per group ) . Representative photograph of M - HFD M - HFD + SCT mice after HFD feeding . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Change of body weight of M-HFD (offspring of dams with high-fat pregnant diets) and M-HFD+SCT (offspring of dams with high-fat pregnant diets supplied with SCT) mice upon HFD feeding (n = 8 per group). Representative photograph of M-HFD and M-HFD+SCT mice after HFD feeding. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


C , Time - resolved oxygen consumption ( C ) in 48 - hour / cycle measured by CLAMS in M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) , phase is marked as background . Regression - absolute oxygen consumption ( C ) of are shown in right part . Data information : Statistical significance was determined using Two - Student ' s t . Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

C, Time-resolved oxygen consumption (C) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute oxygen consumption (C) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel C have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


D Time - resolved heat production ( D ) in 48 - hour / cycle measured by CLAMS in M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) , phase is marked as background . Regression - absolute heat production ( D ) of are shown in right part . Data information : Statistical significance was determined using Two - Student ' s t . Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable , group as fixed variable as covariate . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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D Time-resolved heat production (D) in 48-hour light/dark cycle measured by CLAMS in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group), dark phase is marked as dark background. Regression-based absolute heat production (D) analysis of body weight are shown in the right part. Data information: Statistical significance was determined using Two-tailed Student's t test. Data in panel D have been analyzed using ANCOVA with oxygen consumption or heat production as dependent variable, group as fixed variable and body weight as covariate. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


E GTT ( 1 g glucose / kg ) in M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) AUCs were calculated in right part . Mice were fasted for 18h before assays . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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E GTT (1 g glucose/kg body weight) in M-HFD and M-HFD+SCT mice with HFD (n = 5 per group) and AUCs were calculated in right part. Mice were fasted for 18h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


F ITT ( 0 . 75 U insulin / kg ) in M - HFD M - HFD + SCT mice with HFD , AUCs were calculated in bottom ( n = 5 per group ) . Mice were fasted for 6h before assays . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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F ITT (0.75 U insulin/kg body weight) in M-HFD and M-HFD+SCT mice with HFD, and AUCs were calculated in bottom (n = 5 per group). Mice were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


G Serum TG , TC NEFA in M - HFD M - HFD + SCT mice with HFD . ( n = 8 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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G Serum TG, TC and NEFA levels in M-HFD and M-HFD+SCT mice with HFD. (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


H Representative HE UCP1 of iWAT from M - HFD M - HFD + SCT mice with HFD . Scale bar , 100 μm ( n = 5 per group ) .

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H Representative HE staining and UCP1 staining of iWAT from M-HFD and M-HFD+SCT mice with HFD. Scale bar, 100 μm (n = 5 per group).


I Relative mRNA of UCP1 , PGC1α , PRDM16 , CIDEA DIO2 in iWAT from M - HFD M - HFD + SCT mice with HFD ( n = 5 per group ) . Data information : Statistical significance was determined using Two - Student ' s t . Data are presented as mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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I Relative mRNA levels of UCP1, PGC1α, PRDM16, CIDEA and DIO2 in iWAT from M-HFD and M-HFD+SCT mice with HFD (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.


( F ) 5 - day - old Col - 0Cas9 WT CRISPR glv seedlings were treated with 100 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh . Shown is mean of relative fresh compared to MS medium control - / + SD , n = 30 - 32 biological replicates from 2 pooled experiments ( one - way ANOVA , Tukey post - hoc , a - b p < 0 . 001 ) .

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(F) 5-day-old Col-0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=30-32 biological replicates from 2 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001).


( G ) Ethylene production was measured in Col - 0Cas9 WT CRISPR glv 3 . 5 h after elicitation with 500 nM flg22 or co - treatment with 1 µM GLV2 . Ethylene production was normalized to average of WT responses to flg22 set as 1 . Shown is mean of n = 11 - 12 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc , a - b p < 0 . 01 ) .

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(G) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01).


( H ) Mean cfu of Pto DC3000 COR - 4 days after spray inoculation of Col - 0Cas9 WT CRISPR glv ; n = 12 biological replicates from 3 pooled experiments - / + SD ( Students T - , * p < 0 . 05 ) .

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(H) Mean cfu of Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).


( B ) ROS burst in WT after elicitation with 10 nM flg22 , 1 μM GLV2 , co - treatment or pre - treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22 . Shown is mean of total ROS production ( Total RLU ) over 40 minutes normalized to flg22 response set as 100 % - / + SD , n = 96 - 120 biological replicates from 15 pooled experiments ( one - way ANOVA , Tukey post - hoc , a - b / c / d , p < 0 . 001 ; b - c , p < 0 . 05 ; b - d , p < 0 .

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(B) ROS burst in WT after elicitation with 10 nM flg22, 1 μM GLV2, co-treatment or pre-treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22. Shown is the mean of total ROS production (Total RLU) over 40 minutes normalized to the flg22 response set as 100% -/+ SD, n=96-120 biological replicates from 15 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b/c/d, p<0.001; b-c, p<0.05; b-d, p<0.001


( D ) PR1 expression was measured in 10 - day - old WT seedlings treated for 24 h with 100 nM flg22 , 1 μM GLV2 or co - treatment . Expression was normalized to UBQ house - keeping gene . Shown is mean of n = 4 . Symbol colors represent different biological

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(D) PR1 expression was measured in 10-day-old WT seedlings treated for 24 h with 100 nM flg22, 1 μM GLV2 or co-treatment. Expression was normalized to the UBQ house-keeping gene. Shown is the mean of n=4. Symbol colors represent different biological replicates


( B ) Ethylene production was measured in indicated genotypes 3 . 5 h after elicitation with mock or 500 nM flg22 . Ethylene accumulation was normalized to average of WT responses to flg22 set as 1 . Shown is mean of n = 18 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc , a - b p < 0 . 001 ; a - c , p < 0 . 001 ; b - c , p < 0 . 001 ) .

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(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM flg22. Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).


( E ) Ethylene production was measured in WT rgi5x rgi5x pRGI3 : : RGI3 # 2 # 3 3 . 5 h after elicitation with 500 nM flg22 . Ethylene production was normalized to average of WT responses to flg22 set as 1 . Shown is mean of n = 8 biological replicates from 2 pooled experiments - / + SD ( one - way ANOVA , Dunnett post - hoc , a - b p < 0 . 05 ) .

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(E) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3::RGI3 #2 and #3 3.5 h after elicitation with 500 nM flg22. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05).


( F ) 5 - day - old seedlings of indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh . Shown is mean of relative fresh compared to MS medium control - / + SD , n = 36 biological replicates from 3 pooled experiments ( Students T - , * * * p < 0 . 001 ) .

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(F) 5-day-old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36 biological replicates from 3 pooled experiments (Students T-test, ***p<0.001).


( C ) 5 - day - old seedlings of WT , rgi5x rgi5x pRGI3 : : RGI3 # 2 # 3 were treated with 10 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh . Shown is mean of relative fresh compared to MS medium control - / + SD , n = 32 biological replicates pooled from 4 experiments ( Students T - , * * p < 0 . 01 ) .

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(C) 5-day-old seedlings of WT, rgi5x and rgi5x pRGI3::RGI3 #2 and #3 were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=32 biological replicates pooled from 4 experiments (Students T-test, **p<0.01).


( D ) Quantitative of synthetic GLV2 or GLV2 - S peptide binding to RGI3ECD using microscale thermophoresis . Shown is mean - / + SD , n = 3 technical replicates .

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(D) Quantitative analysis of synthetic GLV2 or GLV2-S peptide binding to RGI3ECD using microscale thermophoresis. Shown is the mean -/+ SD, n=3 technical replicates.


( E ) Co - immunoprecipitation experiment upon co - expression of BAK1 - HA RGI3 - GFP in N . benthamiana . IP was performed using GFP - TRAP agarose beads . Western blots for detection were probed with α - GFP or α - HA antibodies . Similar results were obtained in 3 independent experiments .

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(E) Co-immunoprecipitation experiment upon co-expression of BAK1-HA and RGI3-GFP in N. benthamiana. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Similar results were obtained in 3 independent experiments.


( C ) Co - immunoprecipitation experiment upon co - expression of FLS2 - HA , BAK1 - HA RGI3 - GFP in N . benthamiana . extraction was performed 30 min after mock , 1 μM flg22 or 1 μM GLV2 treatment . IP was performed using GFP - TRAP agarose beads . Western blots for detection were probed with α - GFP or α - HA antibodies . Data information : Similar results were obtained in 3 independent experiments .

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(C) Co-immunoprecipitation experiment upon co-expression of FLS2-HA, BAK1-HA and RGI3-GFP in N. benthamiana. Protein extraction was performed 30 min after mock, 1 μM flg22 or 1 μM GLV2 treatment. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Data information: Similar results were obtained in 3 independent experiments.


( B ) Quantitative real - time PCR of FLS2 transcripts from 12 - day - old seedlings treated with mock or 1 μM GLV2 for indicated time , n = 3 biological replicates . UBQ was used as a house keeping gene . Students T - revealed no statistical difference for each of different timepoints . Data information : Similar results were obtained in 3 independent experiments .

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(B) Quantitative real-time PCR of FLS2 transcripts from 12-day-old seedlings treated with mock or 1 μM GLV2 for the indicated time, n=3 biological replicates. UBQ was used as a house keeping gene. Students T-test revealed no statistical difference for each of the different timepoints. Data information: Similar results were obtained in 3 independent experiments.


( C ) extraction was performed from 12 - day - old seedlings of indicated genotypes . Western blot was probed with α - FLS2 antibodies . ( D ) extraction was performed from 12 - day - old seedlings after 1 hour treatment with mock or 50 μM MG132 . Western blots were probed with α - FLS2 antibodies . Data information : Similar results were obtained in 3 independent experiments .

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(C) Protein extraction was performed from 12-day-old seedlings of the indicated genotypes. Western blot was probed with α-FLS2 antibodies. (D) Protein extraction was performed from 12-day-old seedlings after 1 hour treatment with mock or 50 μM MG132. Western blots were probed with α-FLS2 antibodies. Data information: Similar results were obtained in 3 independent experiments.


C . Phage displaying specific peptides for mATG8 are identified by phage ELISA on immobilized GST - mATG8s negative control BSA GST . Binding signal is shown as absorbance at 450 nm in arbitrary units [ AU ] .

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C. Phage displaying specific peptides for mATG8 are identified by phage ELISA on immobilized GST-mATG8s and negative control proteins BSA and GST. Binding signal is shown as absorbance at 450 nm in arbitrary units [AU].


D . Pull - down of mTurquoise - peptide fusions from lysates on GST - mATG8s beads followed by immuno - blotting with α - GFP . As loading control , with amido black is shown .

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D. Pull-down of mTurquoise-peptide fusions from cell lysates on GST-mATG8s beads followed by immuno-blotting with α-GFP. As loading control, the protein staining with amido black is shown.


B , C , E . Annexin A4 co - translocation assays of peptide binders EGFP - mATG8s . In comparison to single peptides ( B ) , triplication of peptides ( C ) or introduction of negative charges adjacent to binding motif ( E ) increases co - translocation extent of peptide - sensors with their target mATG8 . Control assays with non - target mATG8 are shown in expanded view figure 2 .

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B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2.


D . Biotinylated octameric peptides representing a single acid walk - through along complete 16mer peptide AS _ 67 AS _ M67 are immobilized on streptavidin . Binding of GST - LC3C fusion - to immobilized peptides is detected by a GST specific antibody ( HRP - conjugate ) shown as absorbance at 450 nm . AU = arbitrary

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D. Biotinylated octameric peptides representing a single amino acid walk-through along the complete 16mer peptide AS_67 and AS_M67 are immobilized on streptavidin. Binding of GST-LC3C fusion-protein to the immobilized peptides is detected by a GST specific antibody (HRP-conjugate) and shown as absorbance at 450 nm. AU=arbitrary unit


F . Translocation efficiency of unmodified peptide AS _ 67 , triplicate AS3 _ 67 charge optimized peptide AS _ M67 is classified as % of that show no translocation ' - ' to very good ' + + + + ' translocation with EGFP - LC3C ; n = total number of analyzed . Data are presented as mean of three biological replicates ± SD .

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F. Translocation efficiency of the unmodified peptide AS_67, triplicate AS3_67 and charge optimized peptide AS_M67 is classified as % of cells that show no translocation '-' to very good '++++' translocation with EGFP-LC3C; n = total number of analyzed cells. Data are presented as mean of three biological replicates ±SD.


A , B . recruitment of mCherry fused p62 derived sensor AS3 _ p62 to autophagosomes positive for overexpressed EGFP - LC3B ( A ) or endogenous LC3B ( B ) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment .

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A, B. The recruitment of the mCherry fused p62 derived sensor AS3_p62 to the autophagosomes positive for overexpressed EGFP-LC3B (A) or endogenous LC3B (B) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment.


C . Recruitment of AS3 _ p62 in Atg5 + / + or Atg5 - / - MEFs to endogenous Lc3b .

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C. Recruitment of AS3_p62 in Atg5+/+ or Atg5-/- MEFs to endogenous Lc3b.


D . Lack of recruitment of p62 - derived sensor in absence of hydrophobic residues of LIR motif ( ΔLIR ) to EGFP - LC3B positive autophagosomes .

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D. Lack of recruitment of the p62-derived sensor in the absence of the hydrophobic core residues of the LIR motif (ΔLIR) to EGFP-LC3B positive autophagosomes.


E , F . ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 .

E, F. ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins.


A - C . of autophagy flux in stable inducible HeLa lines expressing mCherry - fusion of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored in basal conditions ( A ) indirectly via lipidation of LC3B turnover of autophagy receptor p62 .

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A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored in basal conditions (A) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.


A - C . of autophagy flux in stable inducible HeLa lines expressing mCherry - fusion of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored upon starvation ( EBSS ) ( B ) indirectly via lipidation of LC3B turnover of autophagy receptor p62 .

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A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored upon starvation (EBSS) (B) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.


A - C . of autophagy flux in stable inducible HeLa lines expressing mCherry - fusion of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored after autophagy induction by Torin I ( C ) indirectly via lipidation of LC3B turnover of autophagy receptor p62 .

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A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored after autophagy induction by Torin I (C) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.


D . FACS mitophagy flux performed in HeLa stably expressing HA - Parkin , mt - mKeima , a GFP fusion of indicated sensor . were treated with OAQ ( oligomycin , antimycin , Q - VD ) for indicated times analyzed by FACS for lysosomal - positive mt - mKeima ( 561nm ) . average of three independent experiments is presented . Representative FACS data from experiments ( GFP control AS3 _ 67 ) are shown in lower panels . Error bars indicate standard deviation . Significance calculation : 2 - way anova of one - way anova . n . s . = not significant . * * * indicates p - value of 0 . 0001 .

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

D. FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA-Parkin, mt-mKeima, and a GFP fusion of the indicated sensor. Cells were treated with OAQ (oligomycin, antimycin, Q-VD) for indicated times and analyzed by FACS for lysosomal-positive mt-mKeima (561nm). The average of three independent experiments is presented. Representative FACS data from experiments (GFP control and AS3_67) are shown in the lower panels. Error bars indicate standard deviation. Significance calculation: 2-way anova of one-way anova. n.s.= not significant. *** indicates p-value of 0.0001.


A . LC3C specific sensor mCh - AS3 _ 67 is recruited to Salmonella ( SFH2 ) escaping SCV . Salmonella SFH2 is carrying a GFP - reporter gene activated by availability of glucose - 6 - phosphate in host ' s cytosol .

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A. LC3C specific sensor mCh-AS3_67 is recruited to Salmonella (SFH2) escaping SCV. Salmonella SFH2 strain is carrying a GFP-reporter gene activated by availability of glucose-6-phosphate in the host's cytosol.


B . Salmonella with prolonged residence strong activation of GFP - reporter gene ( green arrow ) are not recognized by LC3C specific sensor mCh - AS3 _ 67 .

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B. Salmonella with prolonged cytosolic residence and strong activation of the GFP-reporter gene (green arrow) are not recognized by the LC3C specific sensor mCh-AS3_67.


C . A subpopulation of hyper proliferative Salmonella in cytosol is coated with endogenous LC3C . In contrast , endogenous LC3B is enriched in spots in close proximity to .

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C. A subpopulation of hyper proliferative Salmonella in the cytosol is coated with endogenous LC3C. In contrast, endogenous LC3B is enriched in spots in close proximity to the cytosolic bacteria.


D . Sensitivity of LC3C specific sensor mCh - AS3 _ 67 is increased upon fusion of - tethering FYVE domain or PB1 oligomerization domain .

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D. Sensitivity of LC3C specific sensor mCh-AS3_67 is increased upon fusion of membrane-tethering FYVE domain or PB1 oligomerization domain.


E . Introduction of PB1 domain to mCh - AS3 _ p62 increases its performance in recognizing endogenous LC3B .

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E. Introduction of the PB1 domain to mCh-AS3_p62 increases its performance in recognizing endogenous LC3B.


F . High - performance sensor mCh - PB1 - AS3 _ 67 was transiently expressed in Salmonella infected HeLa to monitor xenophagy events .

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F. High-performance sensor mCh-PB1-AS3_67 was transiently expressed in Salmonella infected HeLa cells to monitor xenophagy events.


A , B . LC3C specific sensor mCh - PB1 - AS3 _ 67 is recruited to a subset of mitochondria undergoing mitophagy . Mitochondria were labeled with MitoTracker Deep Red ( ThermoFisher ) . U2OS were transiently co - transfected for indicated sensor mATG8 . Mitophagy was induced by CCCP treatment over three hours . Lysosomal degradation of mitochondria undergoing mitophagy was blocked by addition of BafilomycinA1 ( Baf ) .

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A,B. LC3C specific sensor mCh-PB1-AS3_67 is recruited to a subset of mitochondria undergoing mitophagy. Mitochondria were labeled with MitoTracker Deep Red (ThermoFisher). U2OS cells were transiently co-transfected for indicated sensor and mATG8 proteins. Mitophagy was induced by CCCP treatment over three hours. Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 (Baf).


C . Upon depolarization , endogenous LC3C is localized to mitochondria labeled with mt - mCherry in U2OS . An example of rare events with nearly complete overlap of signals for endogenous LC3C mitochondria ( upper panel ) a representative image ( lower panel ) is given .

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C. Upon depolarization, endogenous LC3C is localized to mitochondria labeled with mt-mCherry in U2OS cells. An example of rare events with nearly complete overlap of signals for endogenous LC3C and mitochondria (upper panel) and a representative image (lower panel) is given.


D . Overlap of LC3C specific sensor signal with endogenous LC3C endogenous LAMP1 in U2OS induced for mitophagy .

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D. Overlap of LC3C specific sensor signal with endogenous LC3C and endogenous LAMP1 in U2OS cells induced for mitophagy.


( a ) Expression of ATG16L1 in a set of different tissues as detected by RT - PCR ( IEC , intestinal epithelial ) . corresponding β - actin control ( with a 518 - bp amplicon ) is given below .

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(a) Expression of ATG16L1 in a set of different tissues as detected by RT-PCR (IEC, intestinal epithelial cells). The corresponding β-actin control (with a 518-bp amplicon size) is given below.


b ) Amounts of ATG16L1 in colonic mucosal biopsy specimens . ( 15 μg ) from rectal mucosal biopsies of Crohn disease normal controls ( N ) were separated by denaturing SDS - PAGE , transferred onto polyvinylidine fluoride ( PVDF ) membranes probed for presence of ATG16L1 using a specific antibody horseradish peroxidase - coupled secondary antibody . abundant 68 . 2 - kDa band corresponds to predicted molecular of isoform encoded by GenBank AY398617 ( UniProt accession number Q676U5 ) . This major form was used for three - dimensional modeling of ATG16L1 ( Fig . 2 ) . 48 . 5 - kDa band corresponds to GenBank EF079890 , which contains exon 9 ( harboring rs2241880 ) in same reading frame as major form .

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b) Amounts of ATG16L1 in colonic mucosal biopsy specimens. Proteins (15 μg) from rectal mucosal biopsies of Crohn disease patients and normal controls (N) were separated by denaturing SDS-PAGE, transferred onto polyvinylidine fluoride (PVDF) membranes and probed for the presence of ATG16L1 using a specific primary antibody and horseradish peroxidase-coupled secondary antibody. The abundant 68.2-kDa band corresponds to the predicted molecular weight of the isoform encoded by GenBank AY398617 (UniProt accession number Q676U5). This major protein form was used for three-dimensional modeling of the ATG16L1 protein (Fig. 2). The 48.5-kDa band corresponds to GenBank EF079890, which contains exon 9 (harboring rs2241880) in the same reading frame as the major form.


( c - i ) Representative immunohistochemistry results ( out of six normal controls six individuals with Crohn disease ) from colonic mucosal tissue samples from normal controls for genotype AA ( c ) GG ( f ) individuals with Crohn disease for genotype AA ( d ) GG ( g ) are shown to demonstrate localization of ATG16L1 . expression patterns in mononuclear ( h ) intestinal epithelial ( i ) of a Crohn disease sample ( rs2241880AA ) are shown at higher magnification . e shows a Crohn disease sample without antibody , used as a control . Bar represents 10 μm in c - g .

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

(c-i) Representative immunohistochemistry results (out of six normal controls and six individuals with Crohn disease) from colonic mucosal tissue samples from normal controls for genotype AA (c) and GG (f) and individuals with Crohn disease for genotype AA (d) and GG (g) are shown to demonstrate the cellular localization of the ATG16L1 protein. Cellular expression patterns in mononuclear cells (h) and intestinal epithelial cells (i) of a Crohn disease sample (rs2241880AA) are shown at higher magnification. e shows a Crohn disease sample without the primary antibody, used as a control. Bar represents 10 μm in c-g.


32 β - strands forming an eight - bladed β - propeller are numbered as in Supplementary Note . location of variant acid T300A in strand β3 , corresponding to rs2241880 , is marked in yellow .

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The 32 β-strands forming an eight-bladed β-propeller are numbered as in Supplementary Note. The location of the variant amino acid T300A in strand β3, corresponding to rs2241880, is marked in yellow.


( A - H ) Six to eight old outbred CD - 1 mice were infected via tail vein injection with 5x107 CFU of WT S . aureus Newman or isogenic mutant strains grown in TSB medium to an OD600 of 0 . 5 . Organs were harvested after ( A - D ) four days or ( E - H ) eight days post - infection , burdens in ( A E ) liver , ( B F ) heart , ( C G ) spleen ( D H ) kidney were determined . Data represent mean ± ( n = 7 biological replicates ) . . * Denotes p < 0 . 05 , * * denotes p < 0 . 005 * * * denotes p < 0 . 0007 , using Mann - Whitney .

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(A-H) Six to eight weeks old female outbred CD-1 mice were infected via tail vein injection with 5x107 CFU of WT S. aureus Newman or isogenic mutant strains grown in TSB medium to an OD600 of 0.5. Organs were harvested after (A-D) four days or (E-H) eight days post-infection, and bacterial burdens in the (A and E) liver, (B and F) heart, (C and G) spleen and (D and H) kidney were determined. Data represent mean ± SEM (n=7 biological replicates).. * Denotes p < 0.05, ** denotes p < 0.005 and *** denotes p < 0.0007, using the Mann-Whitney test.


were grown in MHB - ca medium without or with 20 mM glucose as indicated . ( A ) growth was measured by optical density at 600 nm . Data are representative of five independent experiments .

Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (A) Bacterial growth was measured by optical density at 600 nm. Data are representative of five independent experiments.


were grown in MHB - ca medium without or with 20 mM glucose as indicated . ( B ) PndhC PndhF YFP reporter constructs were used for following gene expression data was normalized by growth . Data are expressed as average ± of three independent experiments . * * * Denotes p < 0 . 001 compared to ndhC expression in WT grown in MHB - ca medium via two - way ANOVA with Tukey ' s posttest .

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Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (B) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth. Data are expressed as average ± SEM of three independent experiments. *** Denotes p < 0.001 compared to ndhC expression in WT strain grown in MHB-ca medium via two-way ANOVA with Tukey's posttest.


( A ) Oxygen consumption of WT isogenic mutant strains was measured with Clark oxygen electrode . were grown in MHB - ca medium until exponential phase ( OD600 = 0 . 6 ) or until late stationary phase ( 24 h ) . were washed with PBS respiration initiated by addition of 20 mM glycerol . Data are expressed as average ± SD of four independent experiments . * * * denotes p < 0 . 001 compared to WT via two - way ANOVA with Tukey ' s posttest .

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(A) Oxygen consumption of WT and isogenic mutant strains was measured with Clark oxygen electrode. Cells were grown in MHB-ca medium until exponential phase (OD600 = 0.6) or until late stationary phase (24 h). Cells were washed with PBS and respiration initiated by the addition of 20 mM glycerol. Data are expressed as average ± SD of four independent experiments. *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.


( B ) NADH / NAD + ratios were measured in grown aerobically in MHB - ca medium for 20 h were measured . 100 % corresponds to a NADH / NAD + of 0 . 003 . Data are expressed as average ± SD of four independent experiments . * Denotes p < 0 . 05 , * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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(B) NADH/NAD+ ratios were measured in cells grown aerobically in MHB-ca medium for 20 h were measured. 100% corresponds to a NADH/NAD+ ratio of 0.003. Data are expressed as average ± SD of four independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


( C ) Acetate was monitored in supernatants of WT isogenic ndh strains at 7 24 h of growth in MHB - ca medium . Data are expressed as average ± SD of three independent experiments . * * Denotes p < 0 . 005 * * * denotes p < 0 . 001 compared to WT via two - way ANOVA with Tukey ' s posttest .

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(C) Acetate concentration was monitored in supernatants of WT and isogenic ndh strains at 7 and 24 h of growth in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.


( D ) MKH2 / MK ratios were measured in grown aerobically for 20 h in MHB - ca medium . Data are expressed as average ± SD of three independent experiments . * Denotes p < 0 . 05 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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(D) MKH2/MK ratios were measured in cells grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.


( A ) carotenoid pigment of different S . aureus strains was extracted by methanol quantified by optical density at 465 nm , values were normalized to WT Newman , which was set at 100 . Data are expressed as average ± SD of five independent experiments . * * Denotes p < 0 . 005 * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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(A) The carotenoid pigment of different S. aureus strains was extracted by methanol and quantified by the optical density at 465 nm, and the values were normalized to WT Newman strain, which was set at 100. Data are expressed as the average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


( B ) Hydrogen peroxide sensitivity was measured by incubating WT isogenic strains , previously grown for 20 h in MHB - ca , with different concentrations of hydrogen peroxide . Serial dilutions were plated in TSB medium for measuring CFU . Data are expressed as average ± SD of three independent experiments .

(B) Hydrogen peroxide sensitivity was measured by incubating WT and isogenic strains, previously grown for 20 h in MHB-ca, with different concentrations of hydrogen peroxide. Serial dilutions were plated in TSB medium for measuring CFU. Data are expressed as the average ± SD of three independent experiments.


( A ) PndhC PndhF YFP reporter constructs were used to follow gene expression under biofilm growth conditions . Data collected after 24 h of static incubation was normalized by optical density at 600 nm . Data are expressed as average ± of three independent experiments . No significant differences were found when ndhC expression in WT was compared via one - way ANOVA with Dunnett ' s posttest .

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(A) PndhC and PndhF YFP reporter constructs were used to follow gene expression under the biofilm growth conditions. Data collected after 24 h of static incubation was normalized by optical density at 600 nm. Data are expressed as average ± SEM of three independent experiments. No significant differences were found when ndhC expression in WT strain was compared via one-way ANOVA with Dunnett's posttest.


( B ) Biofilm formation of Newman WT ndh deficient mutants after static incubation at 37 ° C for 20 h . histogram shows amount of biofilm biomass attached to microtiter plate when were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose 2 % NaCl . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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(B) Biofilm formation of Newman WT and ndh deficient mutants after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


( C ) MKH2 / MK ratios were measured in grown under same conditions as for biofilm formation . Data are expressed as average ± SD of three independent experiments . * Denotes p < 0 . 05 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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(C) MKH2/MK ratios were measured in cells grown under the same conditions as for biofilm formation. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.


( D ) NADH / NAD + ratios were measured in grown under same conditions as for biofilm formation . 100 % corresponds to a NADH / NAD + of 0 . 09 . Data are expressed as average ± SD of four independent experiments performed by duplicates . * Denotes p < 0 . 05 , * * * denotes p < 0 . 001 compared to WT via one - way ANOVA with Dunnett ' s posttest .

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(D) NADH/NAD+ ratios were measured in cells grown under the same conditions as for biofilm formation. 100% corresponds to a NADH/NAD+ ratio of 0.09. Data are expressed as average ± SD of four independent experiments performed by duplicates. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.


Hemolytic activity of Newman WT isogenic mutant strains was measured by incubation of rabbit blood with serially diluted supernatants from 20 h cultures grown in MHB - ca medium without with 25 mM glucose , 40 mM sodium pyruvate or 60 mM sodium acetate . Lysis was monitored by release of hemoglobin . Data are expressed as average ± SD of five independent experiments . * Denotes p < 0 . 05 , * * * denotes p < 0 . 001 , ns denotes no significant compared to WT grown in MHB - ca WT grown in MHB - ca + glucose via two - way ANOVA with Tukey ' s posttest .

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Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium without and with 25 mM glucose, 40 mM sodium pyruvate or 60 mM sodium acetate. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of five independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001, ns denotes no significant compared to WT grown in MHB-ca and WT grown in MHB-ca + glucose via two-way ANOVA with Tukey's posttest.


( A ) Hemolytic activity of Newman WT isogenic mutant strains was measured by incubation of rabbit blood with serially diluted supernatants from 20 h cultures grown in MHB - ca medium . Lysis was monitored by release of hemoglobin . Data are expressed as average ± SD of four independent experiments . * * * Denotes p < 0 . 001 , ns denotes no significant compared to WT via one - way ANOVA with Dunnett ' s posttest .

(A) Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of four independent experiments. *** Denotes p < 0.001, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.


( B ) Biofilm formation of Newman WT isogenic mutant strains after static incubation at 37 ° C for 20 h . histogram shows amount of biofilm biomass attached to microtiter plate when were grown in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose 2 % NaCl . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * Denotes p < 0 . 005 , ns denotes no significant compared to WT via one - way ANOVA with Dunnett ' s posttest .

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(B) Biofilm formation of Newman WT and isogenic mutant strains after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. ** Denotes p < 0.005, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.


( C ) Hemolytic activity of Newman WT isogenic mutant strains grown aerobically for 20 h in MHB - ca medium , in absence or presence of 10 mg / ml FA - deficient BSA 0 . 01 % oleic acid . Data are expressed as average ± SD of five independent experiments . * * Denotes p < 0 . 005 * * * denotes p < 0 . 001 compared to each without any BSA or oleic acid addition via two - way ANOVA with Tukey ' s posttest .

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(C) Hemolytic activity of the Newman WT and isogenic mutant strains grown aerobically for 20 h in MHB-ca medium, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.


( D ) Biofilm formation of Newman WT isogenic mutant strains after static incubation for 20 h in MHB - ca supplemented with 10 % human plasma , 0 . 5 % glucose 2 % NaCl , in absence or presence of 10 mg / ml FA - deficient BSA 0 . 01 % oleic acid . Data are expressed as average ± SD of four independent experiments performed by octuplicates . * * * Denotes p < 0 . 001 compared to each without any BSA or oleic acid addition via two - way ANOVA with Tukey ' s posttest .

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(D) Biofilm formation of Newman WT and isogenic mutant strains after static incubation for 20 h in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.


( E ) Hemolytic activity of Newman ∆ ndhC overexpressing N . gonorrhea acyl - ACP synthetase grown aerobically for 20 h in MHB - ca medium . Data are expressed as average ± SD of four independent experiments . * * Denotes p < 0 . 005 compared to ∆ ndhC with empty plasmid via Student ' s t - .

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(E) Hemolytic activity of Newman ∆ndhC strain overexpressing the N. gonorrhea acyl-ACP synthetase and grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of four independent experiments. ** Denotes p < 0.005 compared to ∆ndhC strain with the empty plasmid via Student's t-test.