Representative flow cytometric of Csf2ra ( CD116 ) expression on different precursors from WT . solid gray histogram represents minus one ( FMO ) controls , open black histogram represents stained samples . gray number represents mean ( MFI ) of FMO controls , black number represents MFI of stained samples . E10 . 5 YS were pooled from 8 - 10 , E14 . 5 lung were pooled from 3 - 4 , E14 . 5 liver were from .

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Representative flow cytometric analysis of Csf2ra (CD116) expression on different fetal precursors from WT embryos. The solid gray histogram represents the fluorescence minus one (FMO) controls, the open black histogram represents stained samples. The gray number represents mean fluorescence intensity (MFI) of FMO controls, the black number represents MFI of stained samples. E10.5 YS cells were pooled from 8-10 embryos, E14.5 lung cells were pooled from 3-4 embryos, and E14.5 liver cells were from individual embryo.


Representative flow cytometric of Csf2ra expressions on pMΦ - Mo - derived AM in Csf2ra - / - recipients 4 after transfer of pMΦ or Mo from E14 . 5 livers . AM from 4 - week old WT mice were included as controls .

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Representative flow cytometric analysis of Csf2ra expressions on pMΦ- and fetal Mo-derived AM in Csf2ra-/- recipients 4 weeks after transfer of pMΦ or fetal Mo from E14.5 fetal livers. AM from 4-week old WT mice were included as controls.


of AM signature - up genes ( D ) , AM signature - down genes ( E ) , AM - specific transcription factors ( F ) in precursors mature AM described above in A . data are from one - seq experiment ( 1 - 2 biological replicates per group ) .

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Heatmap of AM signature-up genes (D), AM signature-down genes (E), and AM-specific transcription factors (F) in the precursors and mature AM described above in A. the data are from one RNA-seq experiment (1 - 2 biological replicates per group).


qPCR ( n = 3 biological replicates ) of Myb , Maf , Mafb , Pparg expression in different precursors from WT mature AM from WT adult mice .

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qPCR (n = 3 biological replicates) analysis of Myb, Maf, Mafb, and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice.


Oxygen consumption rate ( OCR ) of pMΦ Mo from E14 . 5 WT liver at baseline after treatment with oligomycin ( Oligo ) , FCCP or rotenone ( Rot ) antimycin A ( AA ) .

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Oxygen consumption rate (OCR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with oligomycin (Oligo), FCCP or rotenone (Rot) and antimycin A (AA).


Extracellular acidification rate ( ECAR ) of pMΦ Mo from E14 . 5 WT liver at baseline after treatment with glucose , Oligo , or 2 - Deoxy - D - glucose ( 2 - DG ) .

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Extracellular acidification rate (ECAR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with glucose, Oligo, or 2-Deoxy-D-glucose (2-DG).


- seq ( C , n = 2 biological replicates ) of Eno1 , Slc2a6 , Txn1 , Txnip gene expression in pMΦ Mo from E14 . 5 WT liver .

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RNA-seq (C, n = 2 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


qPCR ( D , n = 3 biological replicates ) of Eno1 , Slc2a6 , Txn1 , Txnip gene expression in pMΦ Mo from E14 . 5 WT liver .

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qPCR (D, n = 3 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


C - E . Western blot for CCNA , CCNB , Cdk1 GAPDH ( loading control ) expression in lysates from control ( si - GFP ) p53 - depleted ( si - p53 ; si - p53 _ 1 ) ( C ) MDA - MB - 231 CAL27 lines . Representative are shown .

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C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and p53-depleted (si-p53; si-p53_1) (C) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot for CCNA , CCNB , Cdk1 GAPDH ( loading control ) expression in lysates from control ( si - GFP ) YAP - depleted ( si - YAP ; si - YAP _ 1 ) ( D ) MDA - MB - 231 CAL27 lines . Representative are shown .

C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and YAP-depleted (si-YAP; si-YAP_1) (D) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot for CCNA , CCNB , Cdk1 GAPDH ( loading control ) expression in lysates from control ( si - GFP ) TAZ - depleted ( si - TAZ ) ( E ) MDA - MB - 231 CAL27 lines . Representative are shown .

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C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and TAZ-depleted (si-TAZ) (E) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


A . human breast cancers of METABRIC dataset were stratified according to high or low YAP activity signature [ 47 ] by TP53 mutational status , then of cycline signature score were determined in four groups . Cyclin activity is significantly higher in mut - p53 tumors with high of YAP signature , as visualized by box - plot . Signature scores have been obtained summarizing standardized expression of signature genes into a combined score with zero mean [ 7 ] . values shown in graphs are thus adimensional . bottom top of box are first third quartiles , band inside box is median ; whiskers represent 1st 99th percentiles ; values lower greater are shown as circles ( p < 0 . 0001 , n = 701 ) .

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A. Primary human breast cancers of the METABRIC dataset were stratified according to high or low YAP activity signature [47] and by TP53 mutational status, and then the levels of the cycline signature score were determined in the four groups. Cyclin activity is significantly higher in mut-p53 tumors with high levels of the YAP signature, as visualized by box-plot. Signature scores have been obtained summarizing the standardized expression levels of signature genes into a combined score with zero mean [7]. The values shown in graphs are thus adimensional. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median; whiskers represent 1st and 99th percentiles; values lower and greater are shown as circles (p<0.0001, n=701).


B . Same as in A ) using YAP / TAZ activity signature of Zhang et al . ( 2009 ) ( p < 0 . 0001 , n = 701 ) .

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B. Same as in A) using the YAP/TAZ activity signature of Zhang et al. (2009) (p<0.0001, n=701).


C . Kaplan - Meier representing probability of disease - specific survival in mutant wild - type p53 breast cancer from METABRIC dataset stratified according high or low YAP / TAZ signature score . log - rank p - value reflects significance of association between high of YAP / TAZ signature score shorter survival in in mutant - p53 as compared to wild - type p53 ( p < 0 . 0001 , n = 251 ) . p - values were calculated with two - Student ' s t - .

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C. Kaplan-Meier analysis representing the probability of disease-specific survival in mutant and wild-type p53 breast cancer patients from the METABRIC dataset stratified according high or low YAP/TAZ signature score. The log-rank test p-value reflects the significance of the association between high levels of the YAP/TAZ signature score and shorter survival in in mutant-p53 as compared to wild-type p53 patients (p<0.0001, n=251). All p-values were calculated with two-tailed Student's t-test.


A . SKBr3 MDA - MB - 468 knocked - down as indicated in figure , were transiently transfected with pCCAAT - B2LUC ( 100 ng ) or pmutCCAAT - B2LUC ( 100 ng ) luciferase reporter vectors . Values are means + s . d . of three replicates from three independent experiments .

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A. SKBr3 and MDA-MB-468 cells knocked-down as indicated in the figure, were transiently transfected with pCCAAT-B2LUC (100 ng) or pmutCCAAT-B2LUC (100 ng) luciferase reporter vectors. Values are means + s.d. of three replicates from three independent experiments.


B . RT - qPCR of CCNA , CCNB2 cdk1 mRNA in SKBr3 upon transduction with si - GFP si - YAP ( left graph ) or si - p53 ( right graph ) . Values are means + s . d . of three replicates from three independent experiments .

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B. RT-qPCR of CCNA, CCNB2 and cdk1 mRNA levels in SKBr3 cells upon transduction with si-GFP and si-YAP (left graph) or si-p53 (right graph). Values are means + s.d. of three replicates from three independent experiments.


C . H1299 transfected with indicated siRNAs , were transiently transfected with pCCAAT - B2LUC ( 100 ng ) luciferase reporter vector together with empty pcDNA3 or mutant p53R175H . Representative western blotting to control transfections presented in right panel . Values are means + s . d . of three replicates from three independent experiments .

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C. H1299 transfected with the indicated siRNAs, were transiently transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector together with empty pcDNA3 or mutant p53R175H. Representative western blotting to control the transfections presented in the right panel. Values are means + s.d. of three replicates from three independent experiments.


D . RT - qPCR of CCNA cdk1 mRNA in H1299 upon transfection with si - GFP or si - YAP oligos empty pCDNA3 or mutp53R280H expression vectors as indicated in figure . Error bars represent mean + s . d . , from three biological replicates . P - values are indicated in figures ; two - Student ' s t - was applied throughout .

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D. RT-qPCR of CCNA and cdk1 mRNA levels in H1299 cells upon transfection with si-GFP or si-YAP oligos and empty pCDNA3 or mutp53R280H expression vectors as indicated in the figure. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures; two-tailed Student's t-test was applied throughout.


A B . Coimmunoprecipitation western blot from H1299 lysates showing endogenous YAP ( A ) overexpressed GFP - YAP ( B ) bound to mutp53R273H ( A ) or to mutp53R175H ( B ) . In ( A ) YAP was immunoprecipitated with a rabbit polyclonal antibody rabbit IgG was used as negative control of IP . In ( B ) p53 was immunoprecipitated with a sheep polyclonal antibody total lysate from H1299 transfected with empty pCDNA3 vector was used in as negative control of IP .

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A and B. Coimmunoprecipitation and western blot analysis from H1299 cell lysates showing endogenous YAP (A) and overexpressed GFP-YAP (B) bound to mutp53R273H (A) or to mutp53R175H (B). In (A) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and rabbit IgG was used as negative control of IP. In (B) p53 protein was immunoprecipitated with a sheep polyclonal antibody and the total lysate from H1299 transfected with empty pCDNA3 vector was used in as negative control of IP.


C D . Immunoprecipitations of YAP western blot for p53 binding are performed from lysates of cancer lines expressing different mutant p53 ( described in Materials Methods ) ( C ) YAP was immunoprecipitated with a rabbit polyclonal antibody same amount of rabbit IgG was used as negative control of IP .

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C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.


C D . Immunoprecipitations of YAP western blot for p53 binding are performed from lysates of cancer lines expressing different mutant p53 ( described in Materials Methods ) ( C ) from HCT116 expressing wild type p53 ( D ) . YAP was immunoprecipitated with a rabbit polyclonal antibody same amount of rabbit IgG was used as negative control of IP .

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C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) and from HCT116 cells expressing wild type p53 protein (D). YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.


E . extracts from H1299 transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody . lysates derived from transfected with empty vector were used as negative control of immunoprecipitation .

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E. Cellular extracts from H1299 cells transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody. Cell lysates derived from cells transfected with the empty vector were used as negative control of immunoprecipitation.


F . extracts from MDA - MB - 468 were immunoprecipitated with rabbit polyclonal YAP , sheep polyclonal p53 rabbit polyclonal NF - YB antibodies . Rabbit ( for YAP NF - YB IPs ) sheep ( for p53 IP ) IgGs were used as negative control of IPs .

F. Cellular extracts from MDA-MB-468 were immunoprecipitated with rabbit polyclonal YAP, sheep polyclonal p53 and rabbit polyclonal NF-YB antibodies. Rabbit (for YAP and NF-YB IPs) and sheep (for p53 IP) IgGs were used as negative control of IPs.


G . lysates from CAL27 transfected with si - GFP si - NF - YB oligonucleotides were immunoprecipitated with rabbit polyclonal YAP . As specificity control , immunoprecipitations were performed with rabbit IgG .

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G. Cell lysates from CAL27 cells transfected with si-GFP and si-NF-YB oligonucleotides were immunoprecipitated with rabbit polyclonal YAP. As specificity control, immunoprecipitations were performed with rabbit IgG.


H I . ChIP of mutant p53 , YAP H4 acetylated histone - bound from MDA - MB - 468 on transduction with siRNAs oligos targeting endogenous YAP ( siYAP ) ( H ) , NF - Y ( siNF - YB ) ( I ) , or siGFP as a negative control ( H I ) . experiment was performed in biological triplicates . CCNA , CCNB2 Cdk1 promoter occupancy was analyzed by RT - qPCR . Normalization was performed to amount of input . ChIP results were further normalised on RT - qPCR of a region resulted negative for recruitment of mutant p53 ( Appendix Table S5 ) . P - values were calculated with two Student ' s t - . Statistically significant results were referred with p - value < 0 . 05 .

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H and I. ChIP analysis of mutant p53, YAP and H4 acetylated histone-bound chromatin from MDA-MB-468 cells on transduction with siRNAs oligos targeting endogenous YAP (siYAP) (H), NF-Y (siNF-YB) (I), or siGFP as a negative control (H and I). The experiment was performed in biological triplicates. The CCNA, CCNB2 and Cdk1 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The ChIP results were further normalised on the RT-qPCR of a region resulted negative for the recruitment of mutant p53 (Appendix Table S5). P-values were calculated with two tailed Student's t-test. Statistically significant results were referred with p-value<0.05.


A B . Proliferation curves of SKBr3 knocked - down for YAP ( A ) or p53 ( B ) . si - GFP oligos are used as negative control . viability in A B was determined by trypan blue dye exclusion , in C was determined by ATPlite luminescence . values are means + s . d . of six replicates from three independent experiments .

A and B. Proliferation curves of SKBr3 cell line knocked-down for YAP (A) or p53 (B). si-GFP oligos are used as negative control.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.


C . Viability of CAL27 transfected with si - GFP or si - p53 oligos empty vector or GFP - YAP construct , as indicated in legend to graph . viability in A B was determined by trypan blue dye exclusion , in C was determined by ATPlite luminescence . values are means + s . d . of six replicates from three independent experiments .

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C. Viability of CAL27 cell line transfected with si-GFP or si-p53 oligos and empty vector or GFP-YAP construct, as indicated in the legend to the graph.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.


A . CCNA CCNB2 expression transcripts were analyzed by RT - qPCR in cDNA derived from tumours of control ( saline ) or zoledronic acid ( ZA ) - treated mice ( ref . 31 ) . N = 3 mice per group . P - values were calculated with two - t - .

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A. CCNA and CCNB2 expression transcripts were analyzed by RT-qPCR in cDNA derived from tumours of control (saline) or zoledronic acid (ZA)-treated mice (ref. 31). N=3 mice per group. P-values were calculated with two-tailed t-test.


B . ChIP of mutant p53 - bound from MDA - MB - 231 treated or not with 1 µM cerivastatin ( Cer ) for 24h . CCNB2 promoter occupancy was analyzed by RT - qPCR . Normalization was performed to amount of input . experiment was performed in biological triplicate . P - values were calculated with two - t - .

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B. ChIP analysis of mutant p53 -bound chromatin from MDA-MB-231 cells treated or not with 1 µM cerivastatin (Cer) for 24h. The CCNB2 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The experiment was performed in biological triplicate. P-values were calculated with two-tailed t-test.


C . MDA - MB - 231 treated with indicated concentrations of Cer for 24h were transfected with pCCAAT - B2LUC ( 100 ng ) luciferase reporter vector . Error bars represent mean + s . d . , from three biological replicates . P - values are indicated in figures ; p - values were calculated with two - t - .

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C. MDA-MB-231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures;p-values were calculated with two-tailed t-test.


D . CCNA , CCNB2 Cdk1 expression transcripts were analyzed by RT - qPCR in cDNA derived from MDA - MB - 231 ( left panel ) SKBr3 ( right panel ) lines treated or not with 1 µM Cer for 48h . Error bars represent mean + s . d . , from three biological replicates . P - values were calculated with two - t - . P - value < 0 . 01 .

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D. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231 (left panel) and SKBr3 (right panel) cell lines treated or not with 1 µM Cer for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.


E . Western blot for CCNA , CCNB Actin ( loading control ) expression in lysates from MDA - MB - 231 treated or not with 1 µM Cer .

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E. Western blot analysis for CCNA, CCNB and Actin (loading control) expression in lysates from MDA-MB-231 cells treated or not with 1 µM Cer.


F . CCNA , CCNB2 Cdk1 expression transcripts were analyzed by RT - qPCR in cDNA derived from MDA - MB - 231cell treated or not with 30µM ZA for 48h . Error bars represent mean + s . d . , from three biological replicates . P - values were calculated with two - t - . P - value < 0 . 01 .

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F. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231cell line treated or not with 30µM ZA for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.


G . Viability of siGFP sip53 - MDA - MB - 231 after treatment with increasing amounts of cerivastatin ( 0 , 0 . 01 , 0 . 1 , 1 10 µM ) for 48 h . Data are normalized to untreated . Error bars represent mean + s . d . , from three biological replicates .

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G. Viability assay of siGFP and sip53 -MDA-MB-231 cells after treatment with increasing amounts of cerivastatin (0, 0.01, 0.1, 1 and 10 µM) for 48 h. Data are normalized to untreated. Error bars represent mean + s.d., from three biological replicates.


Mouse YUMM1 . 7 melanoma were subcutaneously inoculated into C57BL / 6 mice when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( C ) Kaplan - Meier survival curves of mice treated with indicated therapies ( n = 6 ) . Log rank ( Mantel - Cox ) statistical was used for MAPKi vs MAPKi / BIBF1120 . * * * * P ≤ 0 . 0001 .

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Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (C) Kaplan-Meier survival curves of mice treated with the indicated therapies (n = 6). Log rank (Mantel-Cox) statistical test was used for MAPKi vs MAPKi/BIBF1120. ****P≤0.0001.


Mouse YUMM1 . 7 melanoma were subcutaneously inoculated into C57BL / 6 mice when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( F ) of collagen thickness ( n = 6 for control , BIBF BRAFi / MEKi groups n = 5 for BRAFi / MEKi + BIBF group ) . Two - way ANOVA statistical was used for statistical of mature collagen thickness . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Significance was calculated against control group . Statistical significance of BIBF vs BIBF + BRAFi / MEKi was also calculated .

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

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (F) Quantification of collagen fibers thickness (n = 6 for control, BIBF and BRAFi/MEKi groups and n = 5 for BRAFi/MEKi + BIBF group). Two-way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification. **P≤0.01, ***P≤0.001, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated.


Mouse YUMM1 . 7 melanoma were subcutaneously inoculated into C57BL / 6 mice when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( G ) showing differential expression of ECM myofibroblast / CAF genes in mice treated with MAPK - targeted therapies with or without BIBF compared to control mice ( log2 , n = 5 ) .

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

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (G) Heatmap showing the differential expression of ECM and myofibroblast/CAF genes in mice treated with MAPK-targeted therapies with or without BIBF compared to control mice (log2 ratio, n = 5).


( H ) Western Blot showing expression of ECM , myofibroblast / CAF phenotype switch markers in human M238R compared to parental M238P . represents mean of expression of 3 independent experiments by RT - qPCR . ( I ) showing expression of ECM , myofibroblast / CAF phenotype switch markers in human M238R treated with BIBF ( 2 µM , 72 h ) or vehicle alone by RT - qPCR ( n = 3 ) .

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

(H) Heatmap and Western Blot showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells compared to the parental M238P cells. Heatmap represents the mean of expression of 3 independent experiments by RT-qPCR. (I) Heatmap showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells treated with BIBF (2 µM, 72 h) or vehicle alone by RT-qPCR (n = 3).


( B ) Relative miRNA expression were quantified in M238P treated for 72 h with BIBF1120 ( BIBF , 2 μM ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) ( 1µM ) , or with MAPKi ( 1 μM ) plus BIBF ( 2 µM ) by RT - qPCR normalized to miR - 16 - 5p . Data is represented as mean ± from a triplicate representative of 3 independent experiments . One - way ANOVA was used for statistical . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . Significance was calculated against control group . Statistical significance of BRAFi / MEKi vs BRAFi / MEKi + BIBF was also calculated . ( C ) Expression of miR - 143 - 3p miR - 145 - 5p in control mice mice treated with indicated therapies was quantified by RT - qPCR . Data is represented as mean ± from 2 independent experiments performed on 6 mice , with 2 sites of injections . One - way ANOVA was used for statistical . * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 . Significance was calculated against control group . Statistical significance of BRAFi / MEKi vs BRAFi / MEKi + BIBF was also calculated .

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

(B) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 (BIBF, 2 μM), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1µM), or with MAPKi (1 μM) plus BIBF (2 µM) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. One-way ANOVA was used for statistical analysis. **P≤0.01, ***P≤0.001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (C) Expression of miR-143-3p and miR-145-5p in control mice and mice treated with the indicated therapies was quantified by RT-qPCR. Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice, with 2 sites of injections. One-way ANOVA was used for statistical analysis. *P≤0.05, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated.


( D ) Relative miRNA expression was quantified in M238P stimulated for 48 h with TGF - β ( 10 ng / mL ) or PDGF - BB ( 20 ng / mL ) by RT - qPCR normalized to miR - 16 - 5p . Data is represented as mean ± from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( E ) Relative miRNA expression was quantified in M238R treated for 48 h with triple kinase inhibitor Nintedanib / BIBF1120 ( BIBF , 2 μM ) , TGF - β receptor kinase inhibitor SB431542 ( SB , 10 µM ) , pan - AKT inhibitor GSK690693 ( GSK , 10 µM ) by RT - qPCR . Data is represented as mean ± from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR 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

(D) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF-β (10 ng/mL) or PDGF-BB (20 ng/mL) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, ***P≤0.001. (E) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib/BIBF1120 (BIBF, 2 μM), the TGF-β receptor kinase inhibitor SB431542 (SB, 10 µM), and the pan-AKT inhibitor GSK690693 (GSK, 10 µM) by RT-qPCR. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, **P≤0.01, ***P≤0.001.


( F , Phenotype switch / invasive / ECM markers were quantified in therapy - naïve ( N ) resistant ( R ) PDX samples . log2 fold change or of fold change R vs N is shown for each couple of samples .

O O O O O O O O O O O O O O O O O O O O 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, Phenotype switch/invasive/ECM markers were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples.


( B ) M238P were treated 72 h with BRAFi ( Vemurafenib , 3 μM ) in presence or absence of LNA - anti - miR - 143 ( LNA - 143 ) or anti - miR - 145 ( LNA - 145 ) ( 50 nM ) or a combination of two . ECM markers RT - qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . One - way ANOVA was used for statistical . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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

(B) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. ECM markers RT-qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001.


( C ) Western Blot of ECM remodeling markers on total lysates from M238P transfected as in ( A ) . ( D ) Western Blot of ECM remodeling markers on total lysates from treated with or without BRAFi indicated combination of

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

(C) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in (A). (D) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of inhibitors


( F ) Migration performed in Boyden chambers . Representative showing migrating in different conditions . histogram represents quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments , using ImageJ software . Paired Student t - was used for statistical . * P ≤ 0 . 05 , * * * 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 O O O O O O O O O O O O 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) Migration assay performed in Boyden chambers. Representative images showing migrating cells in the different conditions. The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. * P≤0.05, ***P≤0.001.


( F ) Luciferase in HEK overexpressing miR - 143 or miR - 145 transfected with a plasmid harboring WT or muted of miR - 143 miR - 145 binding sites present in FSCN1 3 ' UTR . Each bar represents mean ± SE of experiments performed at least in triplicate . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . P - values were calculated using Paired Student 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase assay in HEK cells overexpressing miR-143 or miR-145 transfected with a plasmid harboring the WT or muted sequence of the miR-143 and miR-145 binding sites present in FSCN1 3'UTR. Each bar represents the mean ± SE of experiments performed at least in triplicate. ***P≤0.001, ****P≤0.0001. P-values were calculated using Paired Student t-test.


( G ) RT - qPCR of FSCN1 expression in M238P transfected with indicated mimics . Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments . Paired Student t - was used for statistical . * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) RT-qPCR analysis of FSCN1 expression in M238P cells transfected with the indicated mimics. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ** P≤0.01, ***P≤0.001.


( B ) FSCN1 immunofluorescent of in M238P treated or not with BRAFi ( Vemurafenib , 3 μM ) in presence or absence of LNA - anti - miR - 143 ( LNA - 143 ) or anti - miR - 145 ( LNA - 145 ) ( 50 nM ) or a combination of two . Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments , Mann - Whitney U was used for statistical . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments, Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


M238P were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( D ) Migration performed in Boyden chambers . Representative showing migration of M238P treated with indicated siRNAs . bar graph represents quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments , using ImageJ software . Paired Student t - was used for statistical . * P ≤ 0 . 05 , * * * P ≤ 0 .

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

M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (D) Migration assay performed in Boyden chambers. Representative images showing migration of M238P cells treated with the indicated siRNAs. The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. *P≤0.05, ***P≤0.001


M238P were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( E ) Western Blot of phenotype - switch markers on lysates from M238P transfected with indicated siRNAs .

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

M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Western Blot analysis of phenotype-switch markers on cell lysates from M238P cells transfected with the indicated siRNAs.


BRAFi - resistant M238R overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct . M238R transduced with a Ctrl lentivirus were used as control . ( G ) Western Blot of FSCN1 , phenotype switch markers ECM remodeling markers on lysates from control FSCN1 overexpressing .

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

BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (G) Western Blot analysis of FSCN1, phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells.


BRAFi - resistant M238R overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct . M238R transduced with a Ctrl lentivirus were used as control . ( H ) Crystal violet viability of M238R stably overexpressing FSCN1 during 6 days with indicated doses of BRAFi Vemurafenib . Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . Paired Student t - was used for statistical . * * * * P ≤ 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 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

BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (H) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ****P≤0.0001.


( A , M238P were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . ( B , M238P were treated 72 h with BRAFi ( Vemurafenib , 3 µM ) or a combination of BRAFi ( Vemurafenib , 0 . 5 µM ) MEKi ( Trametinib , 1 µM ) . ( A - B ) of in stained for F - actin ( red ) nuclei ( blue ) . Data is represented as scatter plot with mean ± SD ( n ≥ 30 per condition ) . Mann - Whitney U was used for statistical . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_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 O O O O O O O O O

(A, M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (B, M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (A-B) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


C ) M238P were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . D ) M238P were treated 72 h with BRAFi ( Vemurafenib , 3 µM ) or a combination of BRAFi ( Vemurafenib , 0 . 5 µM ) MEKi ( Trametinib , 1 µM ) . ( C - D ) of focal adhesions number in stained for pPaxillin ( green ) nuclei ( blue ) . Focal adhesions ( FA ) number is represented as mean ± SD ( n ≥ 30 per condition ) . Each point represents average number of focal adhesions per calculated for each field . Paired Student t - has been used for statistical . * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P . ≤ 0 . 0001 . Scale bar 40 μm .

O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O 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) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). D) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (C-D) Images and quantification of focal adhesions number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions (FA) number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test has been used for statistical analysis. *P≤0.01, ***P≤0.001, ****P. ≤0.0001. Scale bar 40 μm.


( E - F ) M238P were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( E ) of in stained for F - actin ( red ) nuclei ( blue ) . Data is represented as scatter plot with mean ± SD ( n ≥ 30 per condition ) . Mann - Whitney U was used for statistical . * * * * P ≤ 0 . 0001 . Scale bar 40 μm . ( F ) of focal adhesions ( FA ) number in stained for pPaxillin ( green ) nuclei ( blue ) . Focal adhesions number is represented as mean ± SD ( n ≥ 30 per condition ) . Each point represents average number of focal adhesions per calculated for each field . Paired Student t - was used for statistical . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (F) Images and quantification of focal adhesions (FA) number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


E ) M238P were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . ( E ) Effect of miR - 143 - 3p or miR - 145 - 5p overexpression on expression of YAP / MRTF target genes assessed by RT - qPCR . Data are normalized to expression in control . Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . Paired Student t - was used for statistical . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (E) Effect of miR-143-3p or miR-145-5p overexpression on the expression of YAP/MRTF target genes assessed by RT-qPCR. Data are normalized to the expression in control cells. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ****P≤0.0001.


M238P were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . Effect of FSCN1 downregulation on MRTFA ( F ) YAP1 ( G ) translocation assessed by immunofluorescence in M238P . Data are represented as scatter plot with mean ± SD ( n ≥ 30 per condition ) . Each point represents / cytoplasm . Mann - Whitney U was used for statistical . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

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

M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). Effect of FSCN1 downregulation on MRTFA (F) and YAP1 (G) nuclear translocation assessed by immunofluorescence in M238P. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm.


M238P were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( H ) RT - qPCR for expression of MRTFA / YAP target genes in M238P transfected with indicated siRNAs . Data are normalized to expression in parental . Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments . Paired Student t - was used for statistical . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . Scale bar 40 μm .

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M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (H) RT-qPCR analysis for the expression of MRTFA/YAP target genes in M238P cells transfected with the indicated siRNAs. Data are normalized to the expression in parental cells. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. Scale bar 40 μm.


( B - C ) Coimmunoprecipitation of Ams1 ( B ) Ape4 ( C ) with Nbr1 . Endogenously TAP - tagged Nbr1 was immunoprecipitated ( IP ) using IgG Sepharose beads . lysates immunoprecipitates were examined by immunoblotting ( IB ) .

(B-C) Coimmunoprecipitation of Ams1 (B) and Ape4 (C) with Nbr1. Endogenously TAP-tagged Nbr1 was immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and immunoprecipitates were examined by immunoblotting (IB).


( D - E ) Subcellular localization of Ams1 ( D ) Ape4 ( E ) . Wild - type ( WT ) , nbr1Δ , sst4Δ , atg1Δ were grown to mid - log phase in EMM medium examined by live imaging . In ( D ) , Ams1 was endogenously tagged at its C - terminus with mCherry . In ( E ) , Ape4 was endogenously tagged at its C - terminus with GFP in same GFP - binding - ( GBP ) fused 3xUb was expressed under P41nmt1 promoter . Cpy1 is a vacuole lumen marker . Scale bar , 3 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Subcellular localization of Ams1 (D) and Ape4 (E). Wild-type (WT), nbr1Δ, sst4Δ, and atg1Δ cells were grown to mid-log phase in EMM medium and examined by live cell imaging. In (D), Ams1 was endogenously tagged at its C-terminus with mCherry. In (E), Ape4 was endogenously tagged at its C-terminus with GFP and in the same cells GFP-binding-protein (GBP) fused 3xUb was expressed under the P41nmt1 promoter. Cpy1 is a vacuole lumen marker. Scale bar, 3 μm.


Localization of Ams1 in WT , lap2Δ , ape2Δ , ape4Δ . Scale bar , 3 μm . Localization of Ape4 in WT , lap2Δ , ape2Δ , ams1Δ . Scale bar , 3 μm .

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

Localization of Ams1 in WT, lap2Δ, ape2Δ, and ape4Δ cells. Scale bar, 3 μm.   Localization of Ape4 in WT, lap2Δ, ape2Δ, and ams1Δ cells. Scale bar, 3 μm.


Ams1 , Lap2 , Ape2 did not co - immunoprecipitate with Ape4 . Endogenously TAP - tagged Ape4 was immunoprecipitated . Ams1 was endogenously tagged with an mCherry tag .

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

Ams1, Lap2, and Ape2 did not co-immunoprecipitate with Ape4. Endogenously TAP-tagged Ape4 was immunoprecipitated. Ams1 was endogenously tagged with an mCherry tag.


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

The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ams1.   The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ape4.


ZZ1 region but not ZZ2 - ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1 . Scale bar , 3 μm . ZZ1 region but not ZZ2 - ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4 . Scale bar , 3 μm . ZZ2 - ZZ3 region but not ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2 . Scale bar , 3 μm . ZZ2 - ZZ3 region but not ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2 . Scale bar , 3 μm .

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

The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1. Scale bar, 3 μm.   The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2. Scale bar, 3 μm.


Surface plasmon resonance ( SPR ) of Nbr1 - Ams1 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using GST Capture Kit . Ams1 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through chip surface .

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

Surface plasmon resonance (SPR) analysis of the Nbr1-Ams1 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ams1 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.


SPR of Nbr1 - Ape4 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using GST Capture Kit . Ape4 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through chip surface .

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

SPR analysis of the Nbr1-Ape4 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ape4 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.


effects of individually mutating first two residues of Ams1 on its Nbr1 - binding ability . Nbr1 - Ams1 interactions were examined by Pil1 co - tethering . Scale bar , 3 μm .

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

The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.


effects of individually mutating third residue other residues in U - shaped loop of Ape4 on its Nbr1 - binding ability . Nbr1 - Ape4 interactions were examined by Pil1 co - tethering . Scale bar , 3 μm .

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

The effects of individually mutating the third residue and other residues in the U-shaped loop of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.


( A - C ) . Mice gain ( A ) , lean mass fat mass ( B ) gWAT index ( C ) . At 8 of , C57BL / 6 mice were switched to HFD . After 12 of HFD feeding , mice were divided into three groups receiving non - exercise , endurance exercise or resistance exercise for 14 days . ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C). Mice body weight gain (A), the lean mass and fat mass (B) and gWAT index (C). At 8 weeks of age, male C57BL/6 mice were switched to HFD. After 12 weeks of HFD feeding, mice were divided into three groups receiving non-exercise, endurance exercise or resistance exercise for 14 days. (n = 8 per group).


( D ) . Relative changes in metabolites in response to resistance exercise . Heat maps show changes of metabolites in serums from mice receiving resistance exercise or non - exercise . C57BL / 6 mice ( 10 ) fed with normal chow were divided into two groups receiving either non - exercise or resistance exercise for 40 min ( n = 6 per group ) . Shades of yellow blue represent fold increase fold decrease of a metabolite , respectively ( see color scale ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Relative changes in metabolites in response to resistance exercise. Heat maps show changes of metabolites in the serums from mice receiving resistance exercise or non-exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group). Shades of yellow and blue represent fold increase and fold decrease of a metabolite, respectively (see color scale).


( G ) . Two Pearson ' s correlation coefficient of plasma AKG level mass index ( BMI ) in Chinese adults ( 10 35 ) .

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

(G). Two tailed Pearson's correlation coefficient analysis of plasma AKG level and body mass index (BMI) in Chinese adults (10 males and 35 females).


( H ) . Serum AKG - time profile obtained before after 40 - min resistant exercise . At 8 of , C57BL / 6 mice were switched to HFD continuously fed with HFD for 12 . At 20 of , mice received resistance exercise for 40 min . Another group of chow fed C57BL / 6 mice ( 10 ) received resistance exercise for 40 min . serum AKG level were tested at 0 , 1 , 2 , 4 6 hrs after exercise ( n = 8 - 10 per group ) .

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 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

(H). Serum AKG concentration-time profile obtained before and after 40-min resistant exercise. At 8 weeks of age, male C57BL/6 mice were switched to HFD and continuously fed with HFD for 12 weeks. At 20 weeks of age, mice received resistance exercise for 40 min. Another group of chow fed male C57BL/6 mice (10 weeks) received resistance exercise for 40 min. The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after exercise (n = 8-10 per group).


( I ) . Serum AKG after exercise . C57BL / 6 mice ( 10 ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1 day or resistance exercise for 40 min . ( n = 8 - 10 per group ) .

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

(I). Serum AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1 day or resistance exercise for 40 min.(n = 8-10 per group).


( J ) . AKG after exercise . C57BL / 6 mice ( 10 ) fed with normal chow were divided into two groups receiving either non - exercise or resistance exercise for 40 min ( n = 6 per group ) .

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

(J). Muscles AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group).


Muscle ICDHm ( K ) , enzyme activity after exercise . C57BL / 6 mice ( 10 ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

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

Muscle ICDHm (K), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


GDH ( L ) , enzyme activity after exercise . C57BL / 6 mice ( 10 ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

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

GDH (L), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


α - KGDH ( M ) enzyme activity after exercise . C57BL / 6 mice ( 10 ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

α-KGDH (M) enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


( A ) . Serum AKG - time profile obtained from C57BL / 6 mice ( 10 ) fed with normal chow before or after AKG gavage ( 10 mg / kg ) . serum AKG level were tested at 0 , 1 , 2 , 4 6 hrs after gavage ( n = 8 per group ) .

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(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after AKG gavage (10 mg/kg). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after gavage (n = 8 per group).


( B - D ) . Water intake ( B ) , cumulative food intake ( C ) gain ( D ) of C57BL / 6 mice . At 12 of , mice were switched to HFD received tap water or water supplemented with 2 % AKG for 11 ( n = 9 per group ) .

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

(B-D). Water intake (B), cumulative food intake (C) and body weight gain (D) of male C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).


Fat lean mass ( E ) of composition from mice after 11 of AKG supplementation ( n = 9 per group ) .

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Fat and lean mass (E) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).


representative ( F ) of composition from mice after 11 of AKG supplementation ( n = 9 per group ) .

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

representative images (F) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( G - I ) . index of BAT ( G ) , gWAT ( H ) iWAT ( I ) from mice after 11 of AKG supplementation ( n = 9 per group ) .

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(G-I). Weight index of BAT (G), gWAT (H) and iWAT (I) from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( J ) . Serum AKG level of mice after 11 of AKG supplementation ( n = 9 per group ) .

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

(J). Serum AKG level of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( K - L ) . Representative ( K ) ( L ) of gWAT iWAT HE from mice after 11 of AKG supplementation ( n = 9 per group ) .

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

(K-L). Representative images (K) and quantification (L) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( M - O ) . Water intake ( M ) , cumulative food intake ( N ) gain ( O ) of C57BL / 6 mice . At 12 of , mice were switched to HFD received tap water or water supplemented with 2 % AKG for 11 ( n = 9 per group ) .

(M-O). Water intake (M), cumulative food intake (N) and body weight gain (O) of female C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).


( P - Q ) . Fat lean mass index ( P ) representative image ( Q ) of composition from mice after 11 of AKG supplementation ( n = 9 per group ) .

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

(P-Q). Fat and lean mass index (P) and representative image (Q) of body composition from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( R - T ) . index of BAT ( R ) , gWAT ( S ) iWAT ( T ) from mice after 11 of AKG supplementation ( n = 9 per group ) .

(R-T). Weight index of BAT (R), gWAT (S) and iWAT (T) from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( U - V ) . Representative ( U ) ( V ) of gWAT iWAT HE from mice after 11 of AKG supplementation ( n = 9 per group ) .

(U-V). Representative images (U) and quantification (V) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( A - B ) . Oxygen consumption in C57BL / 6 mice after 11 of AKG supplementation ( n = 8 per group ) .

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

(A-B). Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).


( C ) . of mice after 11 of AKG supplementation ( n = 9 per group ) .

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(C). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( D - E ) . Representative ( D ) ( E ) of BAT thermogenesis induced by 6 hr cold exposure at 4 ℃ in mice supplemented with AKG for 11 ( n = 9 per group ) .

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(D-E). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group).


mRNA expression of thermogenic genes ( F ) in BAT of mice after 11 of AKG supplementation ( n = 3 - 6 per group ) .

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The mRNA expression of thermogenic genes (F) in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).


Immunoblots ( G ) of UCP1 in BAT of mice after 11 of AKG supplementation ( n = 3 - 6 per group ) .

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Immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).


( H - I ) . DAB ( H ) ( I ) of UCP1 in BAT of mice supplemented with AKG for 11 ( n = 9 per group ) .

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(H-I). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 9 per group).


( J ) . Serum of NEFA in mice supplemented with AKG for 11 ( n = 9 per group ) .

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(J). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks (n = 9 per group).


( K ) . mRNA expression of CD137 , CD40 , TBX1 , TMEM26 , CITED1 slc27a1 in iWAT of mice supplemented with AKG for 11 ( n = 8 per group ) .

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

(K). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group).


( L - M ) . Respiratory exchange ( RER ) in C57BL / 6 mice after 11 of AKG supplementation ( n = 8 per group ) .

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

(L-M). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).


( N - O ) . Immunoblots ( N ) ( O ) of p - HSL ATGL in gWAT of mice after 11 of AKG supplementation ( n = 3 per group ) .

(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group).


( P - Q ) . Representative ( P ) ( Q ) of p - HSL DAB in gWAT iWAT of mice after 11 of AKG supplementation ( n = 9 per group ) .

(P-Q). Representative images (P) and quantification (Q) of p-HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( R ) . mRNA expression of PPARγ , FASN ACC in gWAT iWAT from mice supplemented with AKG for 11 ( n = 6 per group ) .

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

(R). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group).


( S - V ) . Serum of E ( S ) , NE ( T ) , T3 ( U ) , T4 ( V ) in mice supplemented with AKG for 11 ( n = 8 - 9 per group ) .

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(S-V). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group).


( A ) . Serum AKG - time profile obtained from C57BL / 6 mice ( 10 ) fed with normal chow before or after i . p AKG ( 10 mg / kg ) . serum AKG level were tested at 0 , 1 , 2 , 4 6 hrs after injection ( n = 8 per group ) .

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(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group).


( B - C ) . Representative ( B ) ( C ) of BAT thermogenesis after 6 hrs cold exposure at 4 ℃ . C57BL / 6 mice ( 10 ) were i . p . injected with 10 mg / kg AKG or saline immediately exposed to cold stress at 4 ℃ ( n = 8 per group ) .

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(B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hrs cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group).


( D ) . Immunoblots of p - HSL ATGL in gWAT of C57BL / 6 mice ( 10 ) 3 hrs after i . p . injection of 10 mg / kg AKG or saline ( n = 3 per group ) .

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

(D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group).


( E ) . Serum E level in AKG treated mice 3 hrs after i . p . injection ( n = 8 per group ) .

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

(E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group).


Oxygen consumption ( F - G ) in C57BL / 6 mice ( 10 ) i . p . injected with saline , 10 mg / kg AKG , 1 mg / kg SR59230A ( ADRB3 inhibitor ) or AKG + SR59230A ( n = 8 per group ) . injections were performed at 7 : 00 am of second day . Data was summarized in bar graph ( G I ) by or cycle of second day .

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

Oxygen consumption (F-G) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.


RER ( H - I ) in C57BL / 6 mice ( 10 ) i . p . injected with saline , 10 mg / kg AKG , 1 mg / kg SR59230A ( ADRB3 inhibitor ) or AKG + SR59230A ( n = 8 per group ) . injections were performed at 7 : 00 am of second day . Data was summarized in bar graph ( G I ) by or cycle of second day .

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

RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.


( J - N ) . gain ( J ) , cumulative food intake ( K ) , composition ( L ) , fat ( M ) of sham or adrenalectomized C57BL / 6 mice . mice were adrenalectomized at 8 of . Two after surgeries , mice were switched to HFD given free access to tap water or 2 % AKG for 9 ( n = 8 per group ) .

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

(J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).


serum NEFA ( N ) of sham or adrenalectomized C57BL / 6 mice . mice were adrenalectomized at 8 of . Two after surgeries , mice were switched to HFD given free access to tap water or 2 % AKG for 9 ( n = 8 per group ) .

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

serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).


( O - P ) . Representative ( O ) ( P ) of BAT thermogenesis after 6 hrs cold exposure at 4 ℃ in sham or adrenalectomized mice treated with AKG for 9 ( n = 8 per group ) .

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

(O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group).


( Q ) . mRNA expression of themogenic genes in BAT of sham or adrenalectomized mice treated with AKG for 9 ( n = 6 per group ) .

(Q). The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group).


( R - S ) . Immunoblots ( R ) ( S ) of p - HSL ATGL in gWAT of sham or adrenalectomized mice treated with AKG for 9 ( n = 4 per group ) .

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

(R-S). Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).


( T - U ) . Immunoblots ( T ) ( U ) of UCP1 in BAT of sham or adrenalectomized mice treated with AKG for 9 ( n = 4 per group ) .

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

(T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).


( A ) . mRNA expression of AKG - sensing genes in adrenal tissue of 12 - C57BL / 6 mice ( n = 4 per group ) .

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

(A). The mRNA expression of AKG-sensing genes in the adrenal gland tissue of 12-weeks male C57BL/6 mice (n = 4 per group).


( B ) . mRNA expression of OXGR1 in different tissues of 12 - C57BL / 6 mice ( n = 3 per group ) .

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

(B). The mRNA expression of OXGR1 in different tissues of 12-weeks male C57BL/6 mice (n = 3 per group).


( C ) . OXGR1 localization in adrenal medulla indicated by of OXGR1 ( green ) .

(C). OXGR1 localization in adrenal gland medulla indicated by fluorescent staining of OXGR1 (green).


( D ) . mRNA expression of OXGR1 in adrenal of mice 3 hrs after i . p . injection of saline or 10 mg / kg AKG , or immediately after 40 - mins resistance exercise ( n = 8 per group ) .

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

(D). The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i.p. injection of saline or 10 mg/kg AKG, or immediately after 40-mins resistance exercise (n=8 per group).


( E ) . Immunoblots of OXGR1 expression in adrenal chromaffin treated with negative control ( NC ) siRNA or siOXGR1 ( n = 3 per group ) .

(E). Immunoblots and quantification of OXGR1 protein expression in adrenal chromaffin cells treated with negative control (NC) siRNA or siOXGR1 (n = 3 per group).


( F ) . E level in medium from adrenal chromaffin cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 30 mins ( n = 8 per group ) .

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

(F). E level in the medium from adrenal chromaffin cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 30 mins (n = 8 per group).


( G ) . Intracellular calcium ion [ Ca2 + ] changes in adrenal medulla cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 ( n = 30 per group ) .

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

(G). Intracellular calcium ion [Ca2+] changes in adrenal medulla cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 (n = 30 per group).


( H - I ) . gain ( H ) cumulative food intake ( I ) of WT control ( ) or OXGR1 global knock out ( OXGR1KO ) mice . At 12 of , both control KO mice were switched to HFD further divided into two groups , receiving tap water or water supplemented with 2 % AKG for 13 ( n = 8 per group ) .

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

(H-I). Body weight gain (H) and cumulative food intake (I) of male WT control (littermates) or OXGR1 global knock out (OXGR1KO) mice. At 12 weeks of age, both control and KO mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group).


( J - K ) . Representative of composition ( J ) fat lean mass index ( K ) of WT or OXGR1KO mice treated with AKG for 13 ( n = 8 per group ) .

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

(J-K). Representative images of body composition (J) and fat and lean mass index (K) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


( L - M ) . index of iWAT ( L ) gWAT ( M ) in WT or OXGR1KO mice treated with AKG for 13 ( n = 8 per group ) .

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

(L-M). Weight index of iWAT (L) and gWAT (M) in male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


( N - O ) . Immunoblots ( N ) ( O ) of p - HSL ATGL in gWAT of WT or OXGR1KO mice treated with AKG for 13 ( n = 4 per group ) .

(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group).


Oxygen consumption ( P - Q ) of WT or OXGR1KO mice treated with AKG for 13 ( n = 8 per group ) .

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

Oxygen consumption (P-Q) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


RER ( R - S ) of WT or OXGR1KO mice treated with AKG for 13 ( n = 8 per group ) .

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

RER (R-S) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


gain cumulative food intake of OXGR1 adrenal - specific reexpression mice ( OXGR1REAG ) . OXGR1KO mice ( 8 ) were adrenal - specifically injected with control HBAAV2 / 9 - GFP ( OXGR1KO control ) or HBAAV2 / 9 - OXGR1 ( OXGR1REAG ) . Two after injections , mice were switched to HFD further divided into two groups , receiving tap water or water supplemented with 2 % AKG for 12 ( n = 8 per group ) .

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

Body weight gain and cumulative food intake of male OXGR1 adrenal-specific reexpression mice (OXGR1REAG). Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP (OXGR1KO control) or HBAAV2/9-OXGR1 (OXGR1REAG). Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group).


Representative image of composition fat lean mass index of OXGR1REAG mice treated with AKG for 12 ( n = 8 per group ) .

Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


index of gWAT iWAT in OXGR1REAG mice treated with AKG for 12 ( n = 8 per group ) .

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

Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Immunoblots of p - HSL ATGL in gWAT of OXGR1REAG mice treated with AKG for 12 ( n = 8 per group ) .

Immunoblots and quantification of p-HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Oxygen consumption of OXGR1REAG mice treated with AKG for 12 ( n = 8 per group ) .

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

Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


RER of OXGR1REAG mice treated with AKG for 12 ( n = 8 per group ) .

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

RER of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


gain in WT OXGR1KO mice . At 8 of , C57BL / 6 WT control or OXGR1KO mice were switched to HFD . After 12 of HFD feeding , mice were further divided into two groups , receiving non - exercise or resistance exercise for 14 days . ( n = 8 per group ) .

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

Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups, receiving non-exercise or resistance exercise for 14 days. (n = 8 per group).


Exercise - induced loss in WT OXGR1KO mice . weights from exercise mice were subtracted by average of non - exercise control group for each genotype ( n = 8 per group ) .

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

Exercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of non-exercise control group for each genotype (n = 8 per group).


Exercise - induced fat mass loss in WT OXGR1KO mice . Fat mass from exercise mice were subtracted by average fat mass of non - exercise control group for each genotype ( n = 8 per group ) .

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

Exercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice were subtracted by the average fat mass of non-exercise control group for each genotype (n = 8 per group).


Cumulative food intake of WT OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

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

Cumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


index of gWAT ( E ) iWAT ( F ) of OXGR1KO mice after 14 - days resistance exercise ( n = 8 per group ) .

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

Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).


composition of OXGR1KO mice after 14 - days resistance exercise ( n = 8 per group ) .

Body composition of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).


Serum AKG of WT OXGR1KO mice after resistance exercise . WT OXGR1KO mice ( 10 ) fed with normal chow were receiving resistance exercise for 40 min ( n = 8 per group ) . serum AKG were tested before immediately after exercise .

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 B-MEASURED_VAR O O O O O O O

Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise.


Serum E level in OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

Serum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


mRNA expression in BAT of OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

The mRNA expression in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


expression of UCP1 in BAT of OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

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

protein expression of UCP1 in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


mRNA expression of HSL ATGL n gWAT of OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

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

the mRNA expression of HSL and ATGL n the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


Oxygen consumption in OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

Oxygen consumption in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


RER in OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

RER in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


Ingenuity Pathway ( IPA ) of AKG - induced transcriptome signature in adrenal chromaffin treated with AKG . mRAN was extracted from adrenal chromaffin after 3 - hrs incubation of vehicle or AKG ( 100 μM ) . ( n = 3 per group ) .

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

Ingenuity Pathway Analysis (IPA) of AKG-induced transcriptome signature in adrenal chromaffin cell treated with AKG. The mRAN was extracted from adrenal chromaffin cells after 3-hrs incubation of vehicle or AKG (100 μM). (n = 3 per group).


Immunoblots of p - IKK / IKK , p65 , p - IκB / IκB OXGR1 in adrenal chromaffin cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 3 hrs ( n = 3 per group ) .

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

Immunoblots and quantification of p-IKK/IKK, p65, p-IκB/IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group).


p65 translocation in adrenal chromaffin cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 3 hrs ( n = 3 per group ) . Scale bars , 100 μm .

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

p65 translocation in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group). Scale bars, 100 μm.


Immunoblots of p - IKK / IKK , p65 p - IκB / IκB in adrenal chromaffin cultured with vehicle , AKG ( 100 μM ) , IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs .

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

Immunoblots and quantification of p-IKK/IKK, p65 and p-IκB/IκB protein in adrenal chromaffin cells cultured with vehicle, AKG (100 μM), IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs.


E level in medium from adrenal chromaffin cultured with vehicle , AKG ( 100 μM ) , IKK16 or AKG + IKK16 for 3 hrs ( n = 10 per group ) .

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

E level in the medium from adrenal chromaffin cell cultured with vehicle, AKG (100 μM), IKK16 or AKG + IKK16 for 3 hrs (n = 10 per group).


Live DIC DAPI nucleoids of WT DHFR I91L + W133V DHFR strains after being grown at 30 ° C , 37 ° C , or 42 ° C ( I91L + W133V was grown at 40 ° C ) in M9 medium supplemented with for 4 hours ( see Methods ) .

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

Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L+W133V DHFR strains after being grown at 30°C, 37°C, or 42°C (I91L+W133V was grown at 40°C) in M9 medium supplemented with amino acids for 4 hours (see Methods).


Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42 ° C ( for WT , W133V V75H + I155A strains ) or at 40 ° C ( for I91L + W133V V75H + I91L + I155A ) in M9 medium supplemented with . For boxplots in panels , F central band represents median of distribution , box ends represent 25th 75th percentile , whiskers represent 10th 90th percentile , while dots represent 5th 95th percentile . Data was usually obtained from 2 - 3 biological replicates . number of used to derive boxplot distributions in different panels range usually between 200 to 450 ( please refer to Figure 1 source data for exact number of for each dataset ) . See related Figure EV1B .

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Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. For the boxplots in panels , F the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B.


( A ) ( B ) shows abundance of selected nucleotides for mutants I91L + W133V W133V as well as WT treated with 0 . 5μg / ml of Tmp ( Trimethoprim ) after 4 hours of growth at 40 / 42 ° C in M9 minimal medium ( condition of no filamentation ) , of metabolites were normalized to WT at 4 hours when grown under similar conditions . In minimal medium ( B ) Methionine are extremely low of purines ( IMP , AMP ) are also largely rescued with acid supplementation , however dTMP , dTDP dTTP remain extremely low . Error bars represent of at least three biological replicates ( see Methods ) . dashed in each plot represents value of 1 for WT .

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

(A) and (B) shows abundance of selected nucleotides and amino acids for mutants I91L+W133V and W133V as well as WT strain treated with 0.5μg/ml of Tmp (Trimethoprim) after 4 hours of growth at 40/42°C in M9 minimal medium (condition of no filamentation), Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions. In minimal medium (B) Methionine levels are extremely low Levels of purines (IMP, AMP) are also largely rescued with amino acid supplementation, however dTMP, dTDP and dTTP levels remain extremely low. Error bars represent SEM of at least three biological replicates (see Methods). The dashed line in each plot represents value of 1 for WT.


( A ) recA ( B ) recN genes measured by quantitative PCR when WT mutant strains are grown in M9 medium with or without supplementation of or dTMP . WT mutant strains were grown for 4 hours of growth in indicated medium at 37 ° C , while WT treated with different concentrations of Tmp were grown for 4 hours at 42 ° C . Brown bars ( M9 + AA ) in gray shaded correspond to filamentation conditions these are associated with pronounced upregulation of three SOS genes . On other , conditions with loss of filamentation ( with dTMP or no supplementation ) show much less expression . Error bars represent SD of 2 - 3 biological replicates ( see Methods ) .

O O O O 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 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 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

(A) recA (B) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods).


WT were treated with different concentrations of Tmp at 42 ° C for varying amounts of time in acid supplemented M9 medium ( panel B ) or in M9 media supplemented with both GMP ( panel C ) , following which they were spotted on M9 + AA plates allowed to grow at 30 ° C . Colonies were counted next day . In presence of only , there was no loss in viability for any of Tmp ( panel B ) , despite extensive filamentation ( Figure EV1E ) . In contrast , in presence of GMP , showed sharp loss in viability when grown at high Tmp concentrations . In both panels , error bars represent SD of two biological replicates . dashed represents cfu at one - hour timepoint .

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 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 B-CONTROLLED_VAR 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 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

WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint.