Figure 1 ( A ) Waterfall plot showing the percentage of tumor volume change in olaparib - treated tumors compared to the tumor volume on day 1 . + 20 % and - 30 % are marked by doted lines to indicate the range of PR , SD and PD . The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation . TNBC , Triple Negative Breast Cancer ; ER + BC , Estrogen Receptor positive Breast Cancer ; P , primary ; M , metastasis . Error bars indicate SEM from independent tumors ( n ≥ 3 ) . ( B ) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort - 1 . Olaparib - sensitive models are represented with discontinuous lines . Acquisition of PARPi - resistance in PDX302 , STG201 and PDX093 after prolonged exposure to olaparib is shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Waterfall plot showing the percentage of tumor volume change in olaparib-treated tumors compared to the tumor volume on day 1. +20% and -30% are marked by doted lines to indicate the range of PR, SD and PD. The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation. TNBC, Triple Negative Breast Cancer; ER+BC, Estrogen Receptor positive Breast Cancer; P, primary; M, metastasis. Error bars indicate SEM from independent tumors (n ≥ 3).(B) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort-1. Olaparib-sensitive models are represented with discontinuous lines. Acquisition of PARPi-resistance in PDX302, STG201 and PDX093 after prolonged exposure to olaparib is shown.


Figure 2 ( A ) Levels of BRCA1 promoter hypermethylation , levels BRCA1 mRNA and the presence of BRCA1 nuclear foci by immunofluorescence are shown T127 and T162 were used as positive controls for hypermethylated BRCA1 promoter . Error bars indicate SEM from independent tumors ( n ≥ 2 ) . Dashed line indicates mean of BRCA1 mRNA levels in normal breast . PARPi - response is shown in the summary underneath : white box : PD ; black box : PR / CR . Alterations in HRR - related genes in PDX are also indicated . ( B ) Western blot of PALB2 detected in U2OS cells and PDXs . Three biological replicates of PDX093 are shown ; PDX302 is used as PALB2 wild type PDX control . ( C ) YFP - PALB2 recruitment to laser - induced DSBs is impaired in HeLa cells expressing PALB2 p . M296Nfs ( n = 4 , unpaired t - test at 16min ) . Gene - targeting efficiency using Cas9 / mClover - LMNA1 homologous recombination assay of ( D ) siRNA PALB2 cells ( n = 4 , one - way ANOVA ) or ( E ) cells with no PALB2 depletion complemented with wild type and p . M296Nfs siRNA - resistant constructs ( n = 7 , one - way ANOVA ) . Western blots of PALB2 wild type and PALB2 p . M296Nfs for each condition are shown . Error bars indicate SEM from independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Levels of BRCA1 promoter hypermethylation, levels BRCA1 mRNA and the presence of BRCA1 nuclear foci by immunofluorescence are shown T127 and T162 were used as positive controls for hypermethylated BRCA1 promoter. Error bars indicate SEM from independent tumors (n ≥ 2). Dashed line indicates mean of BRCA1 mRNA levels in normal breast. PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Alterations in HRR-related genes in PDX are also indicated.(B) Western blot of PALB2 detected in U2OS cells and PDXs. Three biological replicates of PDX093 are shown; PDX302 is used as PALB2 wild type PDX control.(C) YFP-PALB2 recruitment to laser-induced DSBs is impaired in HeLa cells expressing PALB2 p.M296Nfs (n=4, unpaired t-test at 16min).Gene-targeting efficiency using Cas9/mClover-LMNA1 homologous recombination assay of (D) siRNA PALB2 cells (n=4, one-way ANOVA) or (E) cells with no PALB2 depletion complemented with wild type and p.M296Nfs siRNA-resistant constructs (n=7, one-way ANOVA). Western blots of PALB2 wild type and PALB2 p.M296Nfs for each condition are shown. Error bars indicate SEM from independent experiments.


Figure 3 ( A ) Percentage of geminin - positive , RAD51 nuclear foci - containing cells detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate SEM from independent tumors ( n ≥ 2 ) . PARPi - response is shown in the summary underneath : white box : PD ; black box : PR / CR . Immunofluorescence staining of RAD51 foci in PARPi - and vehicle - treated PDX tumors is shown . Alterations in HRR - related genes are also summarized : hBRCA1 : BRCA1 promoter hypermethylation and lack of BRCA1 expression and BRCA1 nuclear foci formation . ( B ) Restoration of RAD51 foci formation in PARPi acquired - resistant PDXs . Immunofluorescence staining of BRCA1 and RAD51 foci in PARPi - treated tumors from STG201 , PDX302 and the corresponding PARPi acquired - resistant models ( STG201OR and PDX302OR ) . Scale bars : 10µm . ( C ) Quantification of geminin - positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib . Paired t test in PARPi - sensitive ( CR / PR ) vs . PARPi - resistant ( PD ) PDXs .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Immunofluorescence staining of RAD51 foci in PARPi- and vehicle-treated PDX tumors is shown. Alterations in HRR-related genes are also summarized: hBRCA1: BRCA1 promoter hypermethylation and lack of BRCA1 expression and BRCA1 nuclear foci formation.(B) Restoration of RAD51 foci formation in PARPi acquired-resistant PDXs. Immunofluorescence staining of BRCA1 and RAD51 foci in PARPi-treated tumors from STG201, PDX302 and the corresponding PARPi acquired-resistant models (STG201OR and PDX302OR). Scale bars: 10µm.(C) Quantification of geminin-positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib. Paired t test in PARPi-sensitive (CR/PR) vs. PARPi-resistant (PD) PDXs.


Figure 4 ( A ) Percentage of geminin - positive , RAD51 nuclear foci - containing cells in FFPE samples from untreated PDX tumors . Color bars indicate the presence of pathogenic variants in the indicated genes . Error bars indicate SEM from independent tumors ( n ≥ 2 ) . PARPi - response is shown in the summary underneath : black box : PR / CR ; white box : PD . Box colors indicate the PARP inhibitor treatment . Boxes with two colors indicate the same response to both treatments . Olaparib 100mg / kg & 50mg / kg indicates that both doses were tested and resulted in the same response categorization . ( B ) ROC curves of the RAD51 score and HRD score , for PARPi response prediction capacity in the PDX cohort - 2 . Bootstrap statistical test . ( C ) Immunofluorescence staining of 53BP1 and BRCA1 nuclear foci [ with an antibody towards the N - terminus ( B1 - NT ) or C - terminus ( B1 - CT ) of BRCA1 ] in three BRCA1 - mutant , PARPi - resistant models from PDX cohort - 2 . The location of the mutation within the gene is indicated . Scale bars : 10µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells in FFPE samples from untreated PDX tumors. Color bars indicate the presence of pathogenic variants in the indicated genes. Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: black box: PR/CR; white box: PD. Box colors indicate the PARP inhibitor treatment. Boxes with two colors indicate the same response to both treatments. Olaparib 100mg/kg & 50mg/kg indicates that both doses were tested and resulted in the same response categorization.(B) ROC curves of the RAD51 score and HRD score, for PARPi response prediction capacity in the PDX cohort-2. Bootstrap statistical test.(C) Immunofluorescence staining of 53BP1 and BRCA1 nuclear foci [with an antibody towards the N-terminus (B1-NT) or C-terminus (B1-CT) of BRCA1] in three BRCA1-mutant, PARPi-resistant models from PDX cohort-2. The location of the mutation within the gene is indicated. Scale bars: 10µm.


Figure 5 ( B ) Percentage of geminin - positive , RAD51 or γH2AX nuclear foci - containing cells in FFPE tumor samples from patients with HBOC syndrome . The box underneath summarizes the patient ' s young onset ( < 35y : < 35 years ) , her family history ( FH , purple box for PALB2 - related tumors ) and the presence of BRCA1 nuclear foci in the analyzed tumor samples . ( C ) Immunofluorescence staining of γH2AX , BRCA1 and RAD51 foci in three representative FFPE tumors from patients : one RAD51 - and BRCA1 - positive tumor ( Pt03 ) , one RAD51 - and BRCA1 - negative tumor ( Pt11 ) and one PALB2 - related tumor , with BRCA1 but not RAD51 nuclear foci ( Pt20 . 1 ) . Scale bars : 10µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Percentage of geminin-positive, RAD51 or γH2AX nuclear foci-containing cells in FFPE tumor samples from patients with HBOC syndrome. The box underneath summarizes the patient's young onset (<35y: <35 years), her family history (FH, purple box for PALB2-related tumors) and the presence of BRCA1 nuclear foci in the analyzed tumor samples.(C) Immunofluorescence staining of γH2AX, BRCA1 and RAD51 foci in three representative FFPE tumors from patients: one RAD51- and BRCA1-positive tumor (Pt03), one RAD51- and BRCA1-negative tumor (Pt11) and one PALB2-related tumor, with BRCA1 but not RAD51 nuclear foci (Pt20.1). Scale bars: 10µm.


Figure 1 ( A ) The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice ( n = 8 ) and lean controls ( n = 8 ) . WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed a NCD or HFD for 20 weeks . The arrow indicates the time when the high fat diet （ HFD ） starts , and body weight was monitored weekly . ( n = 10 , per genotype ) Representative pictures of adipocyte - specific QKI - deficient mice under NCD or HFD conditions . Gross appearance of tissues from BAT , eWAT and ingWAT fat pads from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice after 20 weeks on the NCD or HFD . H & E staining of BAT , eWAT and ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm ) The gross appearance of BAT , eWAT and ingWAT fat pads after administration of AAV - ShQKI or AAV - NC vectors in local adipose ( ingWAT and BAT ) . The increase in relative body weights following HFD feeding . ( n = 8 , per group ) H & E staining of the liver , BAT and ingWAT from AAV - NC or AAV - ShQKI mice . ( Scale bar represents 50 μm ) .

O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A)The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice (n = 8) and lean controls (n = 8).WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed a NCD or HFD for 20 weeks. The arrow indicates the time when the high fat diet（HFD）starts, and body weight was monitored weekly. (n = 10, per genotype)Representative pictures of adipocyte-specific QKI-deficient mice under NCD or HFD conditions.Gross appearance of tissues from BAT, eWAT and ingWAT fat pads from WT fl/fl and QKI KO fl/fl:AP2-Cre mice after 20 weeks on the NCD or HFD.H&E staining of BAT, eWAT and ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)The gross appearance of BAT, eWAT and ingWAT fat pads after administration of AAV-ShQKI or AAV-NC vectors in local adipose (ingWAT and BAT).The increase in relative body weights following HFD feeding. (n = 8, per group)H&E staining of the liver, BAT and ingWAT from AAV-NC or AAV-ShQKI mice. (Scale bar represents 50 μm).


Figure 2Representative CT images of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on an NCD or HFD , showing the whole - body fat composition . Glucose tolerance tests ( GTT ) and Insulin tolerance tests ( ITT ) were performed on WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . Blood glucose concentrations during GTT and ITT were monitored . ( n = 10 , per genotype ) Blood glucose and plasma insulin levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype ) Plasma Free Fatty Acid ( FFA ) and Glycerol levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype ) H & E and Oil Red O staining , and liver triglyceride ( TG ) content in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm )

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative CT images of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on an NCD or HFD, showing the whole-body fat composition.Glucose tolerance tests (GTT) and Insulin tolerance tests (ITT) were performed on WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. Blood glucose concentrations during GTT and ITT were monitored. (n= 10, per genotype)Blood glucose and plasma insulin levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)Plasma Free Fatty Acid (FFA) and Glycerol levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)H&E and Oil Red O staining, and liver triglyceride (TG) content in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)


Figure 3O2 consumption rates were measured via indirect calorimetry using CLAMS under chow - fed conditions ( n = 8 , per genotype ) . And average light and dark O2 consumption rates are shown on the right . Heat production rates in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice ( n = 8 , per genotype ) . And average light and dark heat production rates are shown on the right . Heatmaps of log2 ( fold change QKI KO fl / fl : AP2 - Cre BAT vs . WT fl / fl BAT ) for genes involved in thermogenesis and oxidation . ( 3 samples , per group ) Photograph of WT fl / fl and QKI KO fl / fl : AP2 - Cre classical BAT in 4 % paraformaldehyde solution , representative transmission electron micrographs , and UCP1 staining of BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice . The average diameter of lipid droplets and the number of mitochondria per field from WT fl / fl and QKI KO fl / fl : AP2 - Cre BAT . ( n = 3 biological replicates ) The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT - PCR . ( n = 4 biological replicates ) Total oxygen consumption rates ( OCR ) per gram in BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions were measured using a Clark electrode . ( n = 3 biological replicates ) The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT - PCR . ( n = 4 biological replicates ) Total oxygen consumption rates ( OCR ) per gram in ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions . ( n = 3 biological replicates ) Representative recording of Continuous measurement of oxygen consumption rate ( OCR ) in isolated mitochondria from BAT of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on NCD ; Oxygen consumption was performed under basal conditions , following the addition of 0 . 25 mg mitochondria ( Mit ) , 5 mM substrate glycerol - 3 - phosphate ( G3P ) , 2 mM GDP , and 450 mM ADP , 2 μg / ml oligomycin , and 1 . 5 μM FCCP . Quantification of experimental data . ( n = 3 biological replicates , per group ) UCP1 - dependent oxygen consumption rates estimated as GDP - inhabitable rates , based on the data shown in ( K ) . ( n = 3 biological replicates )

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3O2 consumption rates were measured via indirect calorimetry using CLAMS under chow-fed conditions (n = 8, per genotype). And average light and dark O2 consumption rates are shown on the right.Heat production rates in WT fl/fl and QKI KO fl/fl:AP2-Cre mice (n = 8, per genotype). And average light and dark heat production rates are shown on the right.Heatmaps of log2 (fold change QKI KO fl/fl:AP2-Cre BAT vs. WT fl/fl BAT) for genes involved in thermogenesis and oxidation. (3 samples, per group)Photograph of WT fl/fl and QKI KO fl/fl:AP2-Cre classical BAT in 4% paraformaldehyde solution, representative transmission electron micrographs, and UCP1 staining of BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice.The average diameter of lipid droplets and the number of mitochondria per field from WT fl/fl and QKI KO fl/fl:AP2-Cre BAT. (n = 3 biological replicates)The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT-PCR. (n = 4 biological replicates)Total oxygen consumption rates (OCR) per gram in BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions were measured using a Clark electrode. (n = 3 biological replicates)The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT-PCR. (n = 4 biological replicates)Total oxygen consumption rates (OCR) per gram in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions. (n = 3 biological replicates)Representative recording of Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from BAT of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on NCD; Oxygen consumption was performed under basal conditions, following the addition of 0. 25 mg mitochondria (Mit), 5 mM substrate glycerol-3-phosphate (G3P), 2 mM GDP, and 450 mM ADP, 2 μg/ml oligomycin, and 1.5 μM FCCP. Quantification of experimental data. (n=3 biological replicates, per group)UCP1-dependent oxygen consumption rates estimated as GDP-inhabitable rates, based on the data shown in (K). (n=3 biological replicates)


Figure 4The protein levels of PGC1α and QKI in siRNA - mediated QKI knockdown differentiated cell , and the quantification graph was shown on the right . ( n = 3 biological replicates ) Immunostaining of PGC1α and UCP1 in NC , Si - QKI 1 , and Si - QKI 2 adipocytes . ( Scale bar represents 20 μm ) . The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates ) The oxygen consumption rates in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates ) Representative pictures of oil red O staining in three adipocyte types : ShScramble + ovCon cells , QKI knockdown cells ( ShQKI + ovCon ) and QKI knockdown with QKI - 5 re - expression cells ( ShQKI + ovQKI ) . The protein levels of PGC1α in these adipocytes , and the quantification graph was shown on the right . ( n = 3 biological replicates ) The relative mRNA levels of genes associated with energy dissipation in ShScramble + ovCon , ShQKI + ovCon and ShQKI + ovQKI differentiated cells . The results represent 3 independent experiments .

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Figure 4The protein levels of PGC1α and QKI in siRNA-mediated QKI knockdown differentiated cell, and the quantification graph was shown on the right. (n=3 biological replicates)Immunostaining of PGC1α and UCP1 in NC, Si-QKI 1, and Si-QKI 2 adipocytes. (Scale bar represents 20 μm).The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)The oxygen consumption rates in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)Representative pictures of oil red O staining in three adipocyte types: ShScramble+ovCon cells, QKI knockdown cells (ShQKI+ovCon) and QKI knockdown with QKI-5 re-expression cells (ShQKI+ovQKI).The protein levels of PGC1α in these adipocytes, and the quantification graph was shown on the right. (n=3 biological replicates)The relative mRNA levels of genes associated with energy dissipation in ShScramble+ovCon, ShQKI+ovCon and ShQKI+ovQKI differentiated cells. The results represent 3 independent experiments.


Figure 5Construction of a series of luciferase reporter vectors ( PGC1α full - length 3 ' UTR T , A , B , C fragments and PGC1α 3 ' UTR QRE motif sites mutant B M1 , B M2 , C M1 , C M2 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3 ' UTR affected by QKI . ( n = 4 biological replicates ) Construction of a series of luciferase reporter vectors ( UCP1 full - length 3 ' UTR T fragment and UCP1 3 ' UTR QRE motif sites mutant T M1 , T M2 , T M3 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3 ' UTR affected by QKI . ( n = 4 biological replicates ) Based on RIP - sequencing data , the distribution of QKI - binding peaks within UCP1 and PGC1α transcript regions . RNA immunoprecipitation followed by real - time PCR . Specific primer for PGC1α 3 ' UTR region 1 , region 2 and UCP1 3 ' UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue , and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP . ( n = 4 biological replicates ) 293T cells were transfected with PGC1α wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of PGC1α293T cells were transfected with UCP1 wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of UCP1 .

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Figure 5Construction of a series of luciferase reporter vectors (PGC1α full-length 3'UTR T, A, B, C fragments and PGC1α 3'UTR QRE motif sites mutant B M1, B M2, C M1, C M2 fragments). Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3'UTR affected by QKI. (n=4 biological replicates)Construction of a series of luciferase reporter vectors (UCP1 full-length 3'UTR T fragment and UCP1 3'UTR QRE motif sites mutant T M1, T M2, T M3 fragments). Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3'UTR affected by QKI. (n=4 biological replicates)Based on RIP-sequencing data, the distribution of QKI-binding peaks within UCP1 and PGC1α transcript regions.RNA immunoprecipitation followed by real-time PCR. Specific primer for PGC1α 3'UTR region 1, region 2 and UCP1 3'UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue, and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP. (n=4 biological replicates)293T cells were transfected with PGC1α wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of PGC1α293T cells were transfected with UCP1 wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of UCP1.


Figure 6The mRNA stability in adipocytes was measured by incubating cells with actinomycin D ( 5 μg / ml ) . Actinomycin D was added to stop transcription , and RNAs were harvested at the indicated times ( x - axis ) after transcription inhibition . Real - time PCR was used to determine remaining UCP1 , PGC1α and Actin mRNA levels compared with the starting time . Increasing PGC1α mRNA half - life by depletion of QKI ( ShScramble T1 / 2 = 4 . 73 h , ShQKI T1 / 2 = 8 . 04 h ) . ( n = 4 biological replicates ) The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers ( GAPDH and Lamin B1 ) to show the purity of fractions . Real - time PCR analysis of mRNA transcript levels of the QKI targets UCP1 and PGC1α in the nuclear and cytoplasmic fractions of differentiated MEF cells transfected with ShScramble or ShQKI . Values represent the cytoplasmic - to - nuclear ratio of each transcript . Actin served as a negative control . ( n = 4 biological replicates ) RNAs from isolated nuclear and cytoplasmic fractionation were harvested after RNA immunoprecipitation ( RIP ) of RNA - protein complexes using either anti - QKI antibody or control IgG . And RIP - RT - PCR showing the PGC1α and UCP1 transcripts majorly interacted with endogenous QKI in the nucleus . ( n = 3 biological replicates ) Control ( ShScramble ) and QKI knockdown ( ShQKI ) adipocyte lysates were subjected to polysome profiling , and a representative polysome profile is shown . Total RNA was extracted from each fraction , and the distribution of UCP1 and PGC1α mRNA was measured via real - time PCR ( K and L ) . The results are shown as the percentage of total mRNA ( % mRNA ) in polysome fractions and represent 3 independent experiments .

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Figure 6The mRNA stability in adipocytes was measured by incubating cells with actinomycin D (5 μg/ml). Actinomycin D was added to stop transcription, and RNAs were harvested at the indicated times (x-axis) after transcription inhibition. Real-time PCR was used to determine remaining UCP1, PGC1α and Actin mRNA levels compared with the starting time. Increasing PGC1α mRNA half-life by depletion of QKI (ShScramble T1/2 = 4.73 h, ShQKI T1/2 = 8.04 h). (n=4 biological replicates)The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers (GAPDH and Lamin B1) to show the purity of fractions.Real-time PCR analysis of mRNA transcript levels of the QKI targets UCP1 and PGC1α in the nuclear and cytoplasmic fractions of differentiated MEF cells transfected with ShScramble or ShQKI. Values represent the cytoplasmic-to-nuclear ratio of each transcript. Actin served as a negative control. (n=4 biological replicates)RNAs from isolated nuclear and cytoplasmic fractionation were harvested after RNA immunoprecipitation (RIP) of RNA-protein complexes using either anti-QKI antibody or control IgG. And RIP-RT-PCR showing the PGC1α and UCP1 transcripts majorly interacted with endogenous QKI in the nucleus. (n=3 biological replicates)Control (ShScramble) and QKI knockdown (ShQKI) adipocyte lysates were subjected to polysome profiling, and a representative polysome profile is shown. Total RNA was extracted from each fraction, and the distribution of UCP1 and PGC1α mRNA was measured via real-time PCR (K and L). The results are shown as the percentage of total mRNA (% mRNA) in polysome fractions and represent 3 independent experiments.


Figure 7The rectal temperature of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice was monitored upon acute cold exposure ( 4 ° C ) . ( n = 10 mice , per group ) H & E staining and UCP1 immunostaining of sections of ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice at RT ( 22 ° C ) or following 72 h of cold exposure ( 4 ° C ) . ( Scale bar : 50 μm ) . The protein levels of UCP1 in ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice at RT or following 72 h of cold exposure ( 4 ° C ) , and quantified protein levels are shown in ( D ) . ( n = 3 biological replicates , per group ) Red Nile staining in BAT injected with Control or QKI knockdown AAV at RT ( 22 ° C ) or following 6 h and 72 h of cold exposure ( 4 ° C ) . TG content in BAT injected with Control or QKI knockdown AAV at RT ( 22 ° C ) or following 6 h and 72 h of cold exposure ( 4 ° C ) . The UCP1 protein level in BAT at RT or following 6 h and 72 h of cold exposure ( 4 ° C ) and the quantified protein levels . ( n = 4 biological replicates , per group ) The mRNA levels of genes related to energy consumption ( PGC1α , UCP1 and Dio2 ) . ( n = 4 biological replicates , per group )

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Figure 7The rectal temperature of WT fl/fl and QKI KO fl/fl:AP2-Cre mice was monitored upon acute cold exposure (4 °C). (n = 10 mice, per group)H&E staining and UCP1 immunostaining of sections of ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT (22 °C) or following 72 h of cold exposure (4 °C). (Scale bar: 50 μm).The protein levels of UCP1 in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT or following 72 h of cold exposure (4 °C), and quantified protein levels are shown in (D). (n=3 biological replicates, per group)Red Nile staining in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).TG content in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).The UCP1 protein level in BAT at RT or following 6 h and 72 h of cold exposure (4 °C) and the quantified protein levels. (n = 4 biological replicates, per group)The mRNA levels of genes related to energy consumption (PGC1α, UCP1 and Dio2). (n=4 biological replicates, per group)


Figure 8The protein levels of QKI in BAT and ingWAT during cold stimulation ( 4 ° C ) and the quantified protein levels . ( n = 3 biological replicates ) The relative luminescence of a QKI promoter reporter under cAMP ( Forskolin and IBMX ) stimulus . ( n = 3 biological replicates ) The effect of transcriptional factors on the QKI promoter , assessed by luciferase activity assays . ( n = 3 biological replicates ) The relative mRNA levels of genes ( UCP1 , PGC1α , Dio2 ) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus , following cAMP ( Forskolin and IBMX ) stimulation . ( n = 3 biological replicates )

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Figure 8The protein levels of QKI in BAT and ingWAT during cold stimulation (4 °C) and the quantified protein levels. (n=3 biological replicates)The relative luminescence of a QKI promoter reporter under cAMP (Forskolin and IBMX) stimulus. (n=3 biological replicates)The effect of transcriptional factors on the QKI promoter, assessed by luciferase activity assays. (n=3 biological replicates)The relative mRNA levels of genes (UCP1, PGC1α, Dio2) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus, following cAMP (Forskolin and IBMX) stimulation. (n=3 biological replicates)


Figure 1 ( A ) Western blot of Beclin ‐ 1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA ‐ Beclin ‐ 1 construct resistant to the siRNA targeting endogenous Beclin ‐ 1 . ( B ) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release . ( C , D ) Quantification of cells in mitosis ( C ) and quantification of each step of mitosis ( D ) by H3Ser10P staining for Control and Beclin ‐ 1 siRNA ‐ treated cells . Error bars represent standard deviations ( s . d . ) ( n = number of counted cells ) calculated on three independent experiments . Two and three asterisks indicate significant results ( P = 0 . 0028 and P = 0 . 0006 , respectively ) . ( E ) Knockdown of Beclin ‐ 1 increases the number of mitotic cells with misaligned chromosomes . siRNA ‐ treated HeLa cells were fixed in PFA , immunolabelled for α ‐ tubulin ( green ) and stained with DAPI ( blue ) . White arrows indicate misaligned chromosomes . Quantification of cells with misaligned chromosomes is shown at the right ( n = number of counted cells ) . Three asterisks indicate significant results ( P = 0 . 0001 ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; HA , haemagglutinin ; PFA , paraformaldehyde ; siRNA , small interfering RNA .

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Figure 1(A) Western blot of Beclin‐1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA‐Beclin‐1 construct resistant to the siRNA targeting endogenous Beclin‐1.(B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release.(C,D) Quantification of cells in mitosis (C) and quantification of each step of mitosis (D) by H3Ser10P staining for Control and Beclin‐1 siRNA‐treated cells. Error bars represent standard deviations (s.d.) (n=number of counted cells) calculated on three independent experiments. Two and three asterisks indicate significant results (P=0.0028 and P=0.0006, respectively).(E) Knockdown of Beclin‐1 increases the number of mitotic cells with misaligned chromosomes. siRNA‐treated HeLa cells were fixed in PFA, immunolabelled for α‐tubulin (green) and stained with DAPI (blue). White arrows indicate misaligned chromosomes. Quantification of cells with misaligned chromosomes is shown at the right (n=number of counted cells). Three asterisks indicate significant results (P=0.0001). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, haemagglutinin; PFA, paraformaldehyde; siRNA, small interfering RNA.


Figure 2Beclin ‐ 1 depletion prolongs prometaphase by delaying chromosome congression . ( A - H ) Time ‐ lapse films of HeLa cells stably expressing YFP ‐ α ‐ tubulin and H2B ‐ Cherry and treated with the indicated siRNA . The pictures are acquired every 10 min . Time lapse starts at nuclear envelope breakdown . ( A ) Mitosis in control cells . ( B ) Western blot of whole ‐ cell lysates from Control , Beclin ‐ 1 and Beclin ‐ 1 + Mad2 siRNA ‐ treated cells . ( C - E ) Three different classes of mitotic progression were observed upon Beclin ‐ 1 depletion . Higher magnification of lagging chromatids in the anaphase of Beclin ‐ 1 ‐ depleted cells is shown on the right of ( D ) . ( F ) The prometaphase delay is dependent on MAD2 and the spindle checkpoint . Cells treated simultaneously with siRNA targeting MAD2 and Beclin ‐ 1 rapidly exit mitosis . ( G ) Average duration of mitosis in different classes of Beclin ‐ 1 ‐ depleted cells , compared to control and MAD2 depletions . n = number of counted cells in three independent experiments . Error bars on graphs represent s . d . ( H ) The frequency of the different Beclin ‐ 1 phenotypes depended on the efficiency of Beclin ‐ 1 knockdown . Mitotic progression in cells treated with increasing concentrations of Beclin ‐ 1 siRNA was followed by video ‐ microscopy . Left panel : western blot of Beclin ‐ 1 upon RNAi treatment ; right panel : quantification of the appearance of the three different mitotic progression patterns , that is , prolonged mitosis with normal anaphase ( as C ) , with lagging chromatids ( as D ) and mitotic exit without clear anaphase ( as E ) upon increased Beclin ‐ 1 knockdown experiments . NEB , nuclear envelop breakdown ; RNAi , RNA interference ; siRNA , small interfering RNA ; YFP , yellow fluorescent protein .

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Figure 2Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression. (A-H) Time‐lapse films of HeLa cells stably expressing YFP‐α‐tubulin and H2B‐Cherry and treated with the indicated siRNA. The pictures are acquired every 10 min. Time lapse starts at nuclear envelope breakdown. (A) Mitosis in control cells. (B) Western blot of whole‐cell lysates from Control, Beclin‐1 and Beclin‐1+Mad2 siRNA‐treated cells. (C-E) Three different classes of mitotic progression were observed upon Beclin‐1 depletion. Higher magnification of lagging chromatids in the anaphase of Beclin‐1‐depleted cells is shown on the right of (D).(F) The prometaphase delay is dependent on MAD2 and the spindle checkpoint. Cells treated simultaneously with siRNA targeting MAD2 and Beclin‐1 rapidly exit mitosis. (G) Average duration of mitosis in different classes of Beclin‐1‐depleted cells, compared to control and MAD2 depletions. n=number of counted cells in three independent experiments. Error bars on graphs represent s.d.(H) The frequency of the different Beclin‐1 phenotypes depended on the efficiency of Beclin‐1 knockdown. Mitotic progression in cells treated with increasing concentrations of Beclin‐1 siRNA was followed by video‐microscopy. Left panel: western blot of Beclin‐1 upon RNAi treatment; right panel: quantification of the appearance of the three different mitotic progression patterns, that is, prolonged mitosis with normal anaphase (as C), with lagging chromatids (as D) and mitotic exit without clear anaphase (as E) upon increased Beclin‐1 knockdown experiments. NEB, nuclear envelop breakdown; RNAi, RNA interference; siRNA, small interfering RNA; YFP, yellow fluorescent protein.


Figure 3 ( A ) Inter ‐ kinetochore distance is reduced in unaligned chromosomes of Beclin ‐ 1 ‐ depleted cells . The distance between each kinetochore pair was quantified in siRNA ‐ treated and control cells treated with nocodazole for 3 h before fixation . Cells were stained with anti ‐ CREST antibody and DAPI . Quantification of the distance between kinetochore pairs ( indicated by coloured bars ) is reported at the bottom and done on sister kinetochores present in the same focal plane . One asterisk indicates significant result ( P = 0 . 0365 ) . Graphs represent three independent experiments ( error bars = s . d . ) . ( B ) Depletion of Beclin ‐ 1 leads to defects in K ‐ MT attachment . siRNA ‐ treated cells were cooled on ice before fixation to reveal stable k ‐ fibres and then stained with anti ‐ α ‐ tubulin ( red ) and anti ‐ CREST ( green ) antibodies . Numbers point to magnified areas and indicate the mode of attachment of k ‐ fibres to kinetochores ( 1 , bi ‐ oriented ; 2 , unattached ; 3 , mono ‐ oriented kinetochores ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; NS , not significant ; siRNA , small interfering RNA .

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Figure 3(A) Inter‐kinetochore distance is reduced in unaligned chromosomes of Beclin‐1‐depleted cells. The distance between each kinetochore pair was quantified in siRNA‐treated and control cells treated with nocodazole for 3 h before fixation. Cells were stained with anti‐CREST antibody and DAPI. Quantification of the distance between kinetochore pairs (indicated by coloured bars) is reported at the bottom and done on sister kinetochores present in the same focal plane. One asterisk indicates significant result (P=0.0365). Graphs represent three independent experiments (error bars=s.d.).(B) Depletion of Beclin‐1 leads to defects in K‐MT attachment. siRNA‐treated cells were cooled on ice before fixation to reveal stable k‐fibres and then stained with anti‐α‐tubulin (red) and anti‐CREST (green) antibodies. Numbers point to magnified areas and indicate the mode of attachment of k‐fibres to kinetochores (1, bi‐oriented; 2, unattached; 3, mono‐oriented kinetochores). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; NS, not significant; siRNA, small interfering RNA.


Figure 4 ( A ) CENP ‐ E and CREST . Two asterisks indicate significant results , P = 0 . 0053 . ( B ) CENP ‐ F and CREST . Three asterisks indicate significant results , P = 0 . 0006 . ( C ) ZW10 and CREST . Three asterisks indicate significant results , P = 0 . 0007 . ( D ) MAD2 and CREST . Two asterisks indicate significant results , P = 0 . 0032 . ( E ) HEC1 and CREST . ( F ) KNL ‐ 1 and CREST . On the right , magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown . Graphs represent three independent experiments ( error bars = s . d . ) . Scale bar , 10 μm . ( G ) Western blot of whole ‐ cell lysates from control and Beclin ‐ 1 siRNA ‐ treated cells . siRNA , small interfering RNA .

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Figure 4(A) CENP‐E and CREST. Two asterisks indicate significant results, P=0.0053.(B) CENP‐F and CREST. Three asterisks indicate significant results, P=0.0006.(C) ZW10 and CREST. Three asterisks indicate significant results, P=0.0007.(D) MAD2 and CREST. Two asterisks indicate significant results, P=0.0032.(E) HEC1 and CREST.(F) KNL‐1 and CREST. On the right, magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown. Graphs represent three independent experiments (error bars=s.d.). Scale bar, 10 μm.(G) Western blot of whole‐cell lysates from control and Beclin‐1 siRNA‐treated cells. siRNA, small interfering RNA.


Figure 5 ( A ) Immunoprecipitation of endogenous Zwint ‐ 1 in HeLa cell extracts expressing HA ‐ Beclin ‐ 1 or HA ‐ GFP . Input is 3 . 5 % of the immunoprecipitated material . ( B ) GST ‐ Zwint ‐ 1 or GST ‐ beads were used to pull down recombinant purified His6 ‐ Beclin ‐ 1 . ( Left ) Coomassie ‐ blue ‐ stained gel of the purified recombinant proteins ( 1 μg ) . ( Right ) A western blot of GST proteins and His6 ‐ Beclin ‐ 1 in the input and bound fractions . His6 ‐ Beclin ‐ 1 in the input represents 4 % of the amount used for the pull ‐ down . ( C ) Zwint ‐ 1 levels do not change in Beclin ‐ 1 ‐ depleted cells . Cells were treated with nocodazole before fixation . On the right , magnified individual kinetochore pairs stained for Zwint ‐ 1 ( red ) and CREST ( green ) , and quantification of Zwint ‐ 1 levels normalized to CREST signal from three independent experiments ( error bars = s . d . ) . ( D ) Beclin ‐ 1 is associated with both the centrosome and the mitotic spindle during mitosis . HeLa cells were permeabilized before fixation and immunostained for Beclin ‐ 1 ( green ) , α ‐ tubulin ( red ) and DNA ( DAPI , blue ) . ( E ) Beclin ‐ 1 is retained on the k ‐ fibres . HeLa cells were cooled on ice before permeabilization and fixation , and stained with anti ‐ α ‐ tubulin ( red ) and anti ‐ Beclin ‐ 1 ( green ) antibodies . ( F ) Beclin ‐ 1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule ‐ dependent manner . Untreated cells ( left ) and nocodazole ‐ treated cells ( right ) were pre ‐ extracted and fixed before labelling with Beclin ‐ 1 ( green ) and Zwint ‐ 1 ( red ) antibodies . ( G ) The fluorescent ‐ tagged Cherry ‐ Beclin ‐ 1 forms foci in the vicinity of the outer kinetochore . HeLa cells transfected with indicated constructs were pre ‐ extracted and fixed before labelling with Zwint ‐ 1 ( green ) antibody . Magnified areas are shown on the right . ( H ) Beclin ‐ 1 localization close to the kinetochore does not depend on the ZW10 protein . Cells treated with Control , Beclin ‐ 1 and ZW10 siRNA were pre ‐ extracted and fixed before labelling with Beclin ‐ 1 ( green ) , CREST ( blue ) and ZW10 ( red ) antibodies . DNA was visualized with DAPI . ( C - H ) Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DNA , deoxyribonucleic acid ; GFP , green fluorescent protein ; GST , glutathione S ‐ transferase ; HA , haemagglutinin .

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Figure 5(A) Immunoprecipitation of endogenous Zwint‐1 in HeLa cell extracts expressing HA‐Beclin‐1 or HA‐GFP. Input is 3.5% of the immunoprecipitated material.(B) GST‐Zwint‐1 or GST‐beads were used to pull down recombinant purified His6‐Beclin‐1. (Left) Coomassie‐blue‐stained gel of the purified recombinant proteins (1 μg). (Right) A western blot of GST proteins and His6‐Beclin‐1 in the input and bound fractions. His6‐Beclin‐1 in the input represents 4% of the amount used for the pull‐down.(C) Zwint‐1 levels do not change in Beclin‐1‐depleted cells. Cells were treated with nocodazole before fixation. On the right, magnified individual kinetochore pairs stained for Zwint‐1 (red) and CREST (green), and quantification of Zwint‐1 levels normalized to CREST signal from three independent experiments (error bars=s.d.).(D) Beclin‐1 is associated with both the centrosome and the mitotic spindle during mitosis. HeLa cells were permeabilized before fixation and immunostained for Beclin‐1 (green), α‐tubulin (red) and DNA (DAPI, blue).(E) Beclin‐1 is retained on the k‐fibres. HeLa cells were cooled on ice before permeabilization and fixation, and stained with anti‐α‐tubulin (red) and anti‐Beclin‐1 (green) antibodies.(F) Beclin‐1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule‐dependent manner. Untreated cells (left) and nocodazole‐treated cells (right) were pre‐extracted and fixed before labelling with Beclin‐1 (green) and Zwint‐1 (red) antibodies.(G) The fluorescent‐tagged Cherry‐Beclin‐1 forms foci in the vicinity of the outer kinetochore. HeLa cells transfected with indicated constructs were pre‐extracted and fixed before labelling with Zwint‐1 (green) antibody. Magnified areas are shown on the right.(H) Beclin‐1 localization close to the kinetochore does not depend on the ZW10 protein. Cells treated with Control, Beclin‐1 and ZW10 siRNA were pre‐extracted and fixed before labelling with Beclin‐1 (green), CREST (blue) and ZW10 (red) antibodies. DNA was visualized with DAPI. (C-H) Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; DNA, deoxyribonucleic acid; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin.


Figure 1Dynamic fluorescence patterns according to cell density in 8 × GTIIC - EmGFP 293T cells . EmGFP intensity in LATS2 knockdown , or forced expression of a constitutively active form of YAP ( YAP - S127A ) . Scale bars indicate 100 μm . Scatter plots of mean frequency of each shRNA per gene . Average of v HOXA4 is indicated as a red dot . Augmented 8 × GTIIC - luciferase reporter activity by loss of HOXA4 . ShSAV1 was used as an experimental control . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction . Quantitative real - time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4Quantitative real - time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression

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Figure 1Dynamic fluorescence patterns according to cell density in 8×GTIIC-EmGFP 293T cells.EmGFP intensity in LATS2 knockdown, or forced expression of a constitutively active form of YAP (YAP-S127A). Scale bars indicate 100 μm.Scatter plots of mean frequency of each shRNA per gene. Average of v HOXA4 is indicated as a red dot.Augmented 8×GTIIC-luciferase reporter activity by loss of HOXA4. ShSAV1 was used as an experimental control. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression vector


Figure 2Quantitative real - time PCR analysis of CTGF , CYR61 , and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4 . ShLATS1 / 2 and shYAP were used as an experimental control . * P < 0 . 05 , * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction . 8 × GTIIC - luciferase reporter assays in HEK 293T cells transfected HOXA4 and / or shLATS1 / 2 . * * P < 0 . 01 vs shCtrl , by two - way ANOVA with Sidak ' s correction . Quantitative real - time PCR analysis of HOXA4 , YAP , CTGF , and CYR61 in HEK 293T cells with overexpression of HOXA4 and / or constitutive active form of YAP ( YAP - 5SA ) . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with LacZ , by ANOVA with Sidak ' s correction . Quantitative real - time PCR analysis of HOXA4 , YAP , CTGF , and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and / or overexpression of YAP - 5SA . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with shCtrl , by ANOVA with Sidak ' s correction .

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Figure 2Quantitative real-time PCR analysis of CTGF, CYR61, and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4. ShLATS1/2 and shYAP were used as an experimental control. *P<0.05, **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.8×GTIIC-luciferase reporter assays in HEK 293T cells transfected HOXA4 and/or shLATS1/2. **P<0.01 vs shCtrl, by two-way ANOVA with Sidak's correction.Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with overexpression of HOXA4 and/or constitutive active form of YAP (YAP -5SA). *P<0.05, **P<0.01 vs YAP-5SA with LacZ, by ANOVA with Sidak's correction.Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and/or overexpression of YAP-5SA. *P<0.05, **P<0.01 vs YAP-5SA with shCtrl, by ANOVA with Sidak's correction.


Figure 3Association of HOXA4 with TEAD1 indicated by co - IP experiments . HEK 293T cells were co - transfected with FLAG - HOXA4 and Myc - TEAD1Association of HOXA4 but not YAP indicated by co - IP experiments . HEK 293T cells were co - transfected with FLAG - HOXA4 and Myc - YAP . Interaction between endogenous TEAD1 and HOXA4 by co - IP experiments in HEK 293T cells . The interaction between endogenous TEAD1 and YAP was used as a positive control . Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1 . Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4 . Arrowheads indicate IgG heavy chain . Arrows indicate TEAD1 ( a . a . 1 - 426 ) . Requirement of TEAD1 ( a . a . 30 - 135 ) for its interaction with HOXA4 via its homeodomain ( a . a . 216 - 274 ) . Failure of TEAD1 ( a . a . 30 - 102 ) , TEA domain alone , to interact with HOXA4 .

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Figure 3Association of HOXA4 with TEAD1 indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-TEAD1Association of HOXA4 but not YAP indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-YAP.Interaction between endogenous TEAD1 and HOXA4 by co-IP experiments in HEK 293T cells. The interaction between endogenous TEAD1 and YAP was used as a positive control.Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1.Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4. Arrowheads indicate IgG heavy chain. Arrows indicate TEAD1 (a.a. 1-426).Requirement of TEAD1 (a.a. 30-135) for its interaction with HOXA4 via its homeodomain (a.a. 216-274).Failure of TEAD1 (a.a. 30-102), TEA domain alone, to interact with HOXA4.


Figure 4ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4 . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Data are expressed as the means ± SEM of three independent experiments . Competitive co - IP assay and densitometry showing significantly reduced TEAD - YAP interaction by overexpression of HOXA4 in a dose - dependent manner in HEK 293T cells . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments .

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Figure 4ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.Competitive co-IP assay and densitometry showing significantly reduced TEAD-YAP interaction by overexpression of HOXA4 in a dose-dependent manner in HEK 293T cells. *P<0.05, **P<0.01, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Figure 5Expression of Hoxa4 in various mouse tissues using qRT - PCR . Expression of glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) mRNA was used as an internal control . WAT ; white adipose tissue , BAT ; brown adipose tissue , Skm ; skeletal muscle . Quantitative real - time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF - BB stimulation for 24 hours . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Quantitative real - time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP . Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP / TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 . Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 . Quantitative real - time PCR analysis of YAP / TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP . The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Proliferation assay in VSMCs transduced with the indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cells were collected and counted at each time point as indicated . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction . BrdU incorporation assays in VSMCs transduced with the indicated genes . The percentage of BrdU positive cells in each group is shown . Nine to twelve images were randomly acquired and analyzed . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction . Proliferation assay in VSMCs transduced with the indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cells were collected and counted at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . BrdU incorporation assays in VSMCs transduced with the indicated genes . The percentage of BrdU positive cells in each group is shown . Nine to twelve images were randomly acquired and analyzed . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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Figure 5Expression of Hoxa4 in various mouse tissues using qRT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal control. WAT; white adipose tissue, BAT; brown adipose tissue, Skm; skeletal muscle.Quantitative real-time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF-BB stimulation for 24 hours. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.Quantitative real-time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP/TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.Quantitative real-time PCR analysis of YAP/TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction.BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Figure 6Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1 / 3 / 4 . Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction . Proliferation assay in VSMCs transduced with indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cell viability was analyzed by Cell Counting Kit - 8 at indicated days at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments . Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co - IP experiments in human VSMCs with knockdown of HOXA4 . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments . ChIP assays of CTGF , CYR61 and α - SMA promoters using anti - FLAG - tag , anti - Myc - tag , anti - TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc - tagged YAP - 5SA and / or FLAG - tagged HOXA4 . * P < 0 . 05 , * * P < 0 . 01 vs FLAG - GFP , by unpaired two - tailed Student ' s t - test . Data are expressed as the means ± SEM of three independent experiments .

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Figure 6Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P<0.05, **P<0.01, by ANOVA with Sidak's correction.Proliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.ChIP assays of CTGF, CYR61 and α-SMA promoters using anti-FLAG-tag, anti-Myc-tag, anti-TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc-tagged YAP-5SA and/or FLAG-tagged HOXA4. *P<0.05, **P<0.01 vs FLAG-GFP, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.


Figure 7Quantitative real - time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8 - week - old WT and Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) . Quantitative real - time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at the baseline was defined as 1 . 0 . Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation ( n = 6 ) . Quantitative real - time PCR analysis of Yap / Tead target genes in the left carotid artery of 8 - week - old WT and Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) . Quantitative real - time PCR analysis of Yap / Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at the baseline was defined as 1 . 0 . Representative western blotting analysis and densitometry of non - ligated ( right ; R ) and ligated ( left ; L ) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation ( WT : n = 6 , Hoxa4 KO : n = 7 ) . * P < 0 . 05 , * * P < 0 . 01 , by two - way ANOVA with Sidak ' s correction . Representative images showing Elastica van Gieson staining ( G ) , and vessel , neointima and media area ( H ) , and neointima to media layer ratio ( I ) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation ( WT : n = 14 , Hoxa4 KO : n = 11 ) .

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Figure 7Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation (n=6).Quantitative real-time PCR analysis of Yap/Tead target genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).Quantitative real-time PCR analysis of Yap/Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.Representative western blotting analysis and densitometry of non-ligated (right; R) and ligated (left; L) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation (WT: n=6, Hoxa4 KO: n=7). *P<0.05, **P<0.01, by two-way ANOVA with Sidak's correction.Representative images showing Elastica van Gieson staining (G), and vessel, neointima and media area (H), and neointima to media layer ratio (I) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation (WT: n=14, Hoxa4 KO: n=11).


Figure 1Balb / C mice were aerosolically challenged with Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR strains . Each data point represents log10 CFU obtained from the infected lung of mice and e rror bar depicts the median with interquartile range for each group . Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group .

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Figure 1Balb/C mice were aerosolically challenged with Rv, Rv∆aosR, and Rv∆aosR::aosR strains. Each data point represents log10 CFU obtained from the infected lung of mice and error bar depicts the median with interquartile range for each group. Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group.


Figure 2Each data point represents Log10 CFU / lung of an infected animal and the error bar depicts the median with interquartile range for each group .

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Figure 2Each data point represents Log10 CFU/lung of an infected animal and the error bar depicts the median with interquartile range for each group.


Figure 3Relative gene expression of selected DEGs measured by qRT - PCR in Rv compared with Rv treated with CHP ( g ) and Rv as compared with Rv ∆ aosR under oxidative conditions ( h ) . Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates .

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Figure 3Relative gene expression of selected DEGs measured by qRT-PCR in Rv compared with Rv treated with CHP (g) and Rv as compared with Rv∆aosR under oxidative conditions (h). Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates.


Figure 4WCLs of Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15 % SDS - PAGE , transferred to nitrocellulose membrane , and probed with indicated antibodies . pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p . i ( mean ± SEM ; n = 3 ) .

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Figure 4WCLs of Rv, Rv∆aosR, and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane, and probed with indicated antibodies.pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p.i (mean ± SEM; n=3).


Figure 5The promoter of sigA and mec was fused to luciferase , and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured ( mean ± SD ; n = 6 ) Rv : : pN and Rv : : pN - F - sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross - linking . 1 mg WCL was used to immunoprecipitate FLAG - SigH and 1 / 10th IP was probed with α - FLAG and 9 / 10th IP with α - AosR . 50 µg WCLs were probed with α - FLAG , α - AosR , and α - SigA as controls . Expression of CysO , CysM , AosR and GroEL1 ( control ) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h . 5 - 7 . 5 x 104 end labelled 100 bp double - stranded oligonucleotide was incubated with no protein ( lanes 1 and 6 ) or 4 μΜ BSA ( lanes 2 and 7 ) or 2 μΜ His - AosR + 2 μΜ BSA ( lanes 3 and 8 ) or , 2 μΜ His - SigH + 2 μΜ BSA ( lanes 4 and 9 ) or 2 μΜ His - AosR + 2 μΜ His - SigH ( lanes 5 and 10 ) . The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols , respectively , and resolved on 8 % PAGE gel . Protein - DNA complexes ( bound ) and free probe are indicated by arrows . To confirm the specificity of DNA - protein interaction , EMSA reaction was performed in the presence of 5 , 10 , 25 , 50 , and 100 molar excess of unlabelled 100 bp double - stranded oligonucleotide .

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Figure 5The promoter of sigA and mec was fused to luciferase, and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=6)Rv::pN and Rv::pN-F-sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-AosR. 50 µg WCLs were probed with α-FLAG,α-AosR, and α-SigA as controls.Expression of CysO, CysM, AosR and GroEL1 (control) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h.5-7.5 x 104 end labelled 100 bp double-stranded oligonucleotide was incubated with no protein (lanes 1 and 6) or 4 μΜ BSA (lanes 2 and 7) or 2 μΜ His-AosR + 2 μΜ BSA (lanes 3 and 8) or , 2 μΜ His-SigH + 2 μΜ BSA (lanes 4 and 9) or 2 μΜ His-AosR + 2 μΜ His-SigH (lanes 5 and 10). The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols, respectively, and resolved on 8% PAGE gel. Protein-DNA complexes (bound) and free probe are indicated by arrows. To confirm the specificity of DNA-protein interaction, EMSA reaction was performed in the presence of 5, 10, 25, 50, and 100 molar excess of unlabelled 100 bp double-stranded oligonucleotide.


Figure 6Msm : : FLAG - SigH , Msm : : FLAG - SigH - AosR - HA and Msm : : FLAG - SigH - AosRAxxxA - HA were either subjected to none or 50 µM of CHP for 3 h followed by cross - linking . 1 mg WCL was used to immunoprecipitate FLAG - SigH and 1 / 10th IP was probed with α - FLAG and 9 / 10th IP with α - HA . 50 µg WCLs were probed with α - FLAG , α - HA and α - SigA as controls .

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Figure 6Msm::FLAG-SigH, Msm::FLAG-SigH-AosR-HA and Msm::FLAG-SigH-AosRAxxxA-HA were either subjected to none or 50 µM of CHP for 3 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-HA. 50 µg WCLs were probed with α-FLAG, α-HA and α-SigA as controls.


Figure 7Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified . Data presented is representative of one of two biological replicates , each performed in triplicates ( n = 3 ) . Y - axis represents µM thiol / µg of WCL ( mean ± SD ) . Metabolite levels of reduced mycothiol , MSH ergothioneine , ERG were measured in Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR in normal and CHP stressed cells using LC - MRM MS / MS analysis . The absolute values obtained under normal conditions were normalized to 100 % for each strain and the relative values were calculated for samples processed from CHP treated cells . The data indicates relative levels of metabolites ( mean percent ± SD ) of four replicates ( n = 4 ) . qRT - PCR measurements for the log2 fold induction ( mean ± SD , n = 3 ) for indicated genes in Rv ∆ aosR and Rv ∆ aosR treated with 1mM L - cysteine as compared with Rv . All strains were treated with 50 μM CHP . Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates .

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Figure 7Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified. Data presented is representative of one of two biological replicates, each performed in triplicates (n=3). Y-axis represents µM thiol /µg of WCL (mean ± SD). Metabolite levels of reduced mycothiol, MSH ergothioneine, ERG were measured in Rv, Rv∆aosR, and Rv∆aosR::aosR in normal and CHP stressed cells using LC-MRM MS/MS analysis. The absolute values obtained under normal conditions were normalized to 100% for each strain and the relative values were calculated for samples processed from CHP treated cells. The data indicates relative levels of metabolites (mean percent ± SD) of four replicates (n=4).qRT-PCR measurements for the log2 fold induction (mean ±SD, n=3) for indicated genes in Rv∆aosR and Rv∆aosR treated with 1mM L-cysteine as compared with Rv. All strains were treated with 50 μM CHP. Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates.


Figure 1 ( B ) Western blot analysis of lysates and media from wt and mutant N9 cells ( N9 wt / mu ) using antibody anti - murine TREM2 ( clone 5F4 ) , which is raised against the murine TREM2 extracellular domain . sTREM2 , soluble TREM2 * Indicate unspecific bands . Anti - calnexin antibody was used as loading control . ( C ) Phagocytosis of 1 µM HiLyte ™ Fluor 488 Aβ1 - 42 ( fAβ42 ) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non - binding antibody 6687 . Cytochalasin D ( CytoD , 10 mM ) was used as control to verify phagocytic uptake . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 61 , genotype P < 0 . 0001 , treatment P = 0 . 0001 ; post hoc tests wt vs . mu for the following conditions : fAβ42 P = 0 . 0043 , fAβ42 - 2D8 P = 0 . 0436 ) . ( D ) Western blot of BMDM derived from wt and Trem2 knockout ( ko ) animals using antibody 5F4 . ( E ) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8 , or the non - binding control antibody 6687 . ( n = 3 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0005 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 P = 0 . 0021 , fAβ42 - 2D8 1 µg / ml P < 0 . 0001 , fAβ42 - 2D8 5 µg / ml P < 0 . 0001 , fAβ42 - 2D8 10 µg / ml P < 0 . 0001 , fAβ42 / 6687 10 µg / ml P = 0 . 0007 ) ( F ) Quantification of relative fAβ42uptake to lowest antibody concentration used . ( G ) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0223 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 - mAb11 1 µg / ml P = 0 . 0391 , fAβ42 - mAb11 5 µg / ml P = 0 . 0069 , fAβ42 - mAb11 10 µg / ml P < 0 . 0001 , fAβ42 - mAb11 20 µg / ml P = 0 . 0001 , fAβ42 - mAb11 50 µg / ml P < 0 . 0001 ) . ( H ) Quantification of relative fAβ42 uptake to lowest antibody concentration used . ( I ) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2 - deficient BMDM . Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 ( 10 µg / ml ) . ( K ) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 5 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 4797 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 - mAb11 5 µg / ml P = 0 . 0449 , fAβ42 - mAb11 10 µg / ml P = 0 . 0370 , fAβ42 - mAb11 20 µg / ml P = 0 . 0299 , fAβ42 - mAb11 50 µg / ml P = 0 . 0120 ) . ( L ) Quantification of relative fAβ42uptake to lowest antibody concentration used .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Western blot analysis of lysates and media from wt and mutant N9 cells (N9 wt /mu) using antibody anti-murine TREM2 (clone 5F4), which is raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2* Indicate unspecific bands. Anti-calnexin antibody was used as loading control.(C) Phagocytosis of 1 µM HiLyte™ Fluor 488 Aβ1-42 (fAβ42) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non-binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.61, genotype P<0.0001, treatment P=0.0001; post hoc tests wt vs. mu for the following conditions: fAβ42 P=0.0043, fAβ42-2D8 P=0.0436).(D) Western blot of BMDM derived from wt and Trem2 knockout (ko) animals using antibody 5F4.(E) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8, or the non-binding control antibody 6687. (n = 3, +/- SEM; 2 way ANOVA, interaction P=0.0005, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42 P=0.0021, fAβ42-2D8 1 µg/ml P<0.0001, fAβ42-2D8 5 µg/ml P<0.0001, fAβ42-2D8 10 µg/ml P<0.0001, fAβ42/6687 10 µg/ml P=0.0007)(F) Quantification of relative fAβ42uptake to lowest antibody concentration used.(G) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0223, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 1 µg/ml P=0.0391, fAβ42-mAb11 5 µg/ml P=0.0069, fAβ42-mAb11 10 µg/ml P<0.0001, fAβ42-mAb11 20 µg/ml P=0.0001, fAβ42-mAb11 50 µg/ml P<0.0001).(H) Quantification of relative fAβ42 uptake to lowest antibody concentration used.(I) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2-deficient BMDM. Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 (10 µg/ml).(K) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.4797, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 5 µg/ml P=0.0449, fAβ42-mAb11 10 µg/ml P=0.0370, fAβ42-mAb11 20 µg/ml P=0.0299, fAβ42-mAb11 50 µg/ml P=0.0120).(L) Quantification of relative fAβ42uptake to lowest antibody concentration used.


Figure 2 ( A ) Representative histograms for Fcγ - receptors - PE ( FcγR - PE ) expression levels as used for quantification . Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR ( I , IIB / III and IV ) . ( B ) Relative quantification of cell surface levels of FcγR molecules . Absolute number of cell surface FcγR - PE molecules was determined by the BD QuantiBRITE ® method ( see methods section for details ) and normalized to expression levels of the respective wt control . ( n = 4 , + / - SEM , t test , two - tailed ; wt vs . ko : FcγRI - PE P = 0 . 0008 , FcγRIIB / III - PE P = 0 . 0033 , FcγRIV - PE P = 0 . 7001 ) . ( C ) mRNA levels of FcγR are increased in Trem2 ko BMDM . Fold changes of the respective FcγR ( I , IIB , III , IV ) mRNA levels in Trem2 ko BMDM were determined by quantitative real - time PCR . ( n = 6 , + / - SEM ; one - sample t test , two - tailed ; wt vs . ko : FcγRI P = 0 . 0118 , FcγRIIB P = 0 . 0006 , FcγRIII P = 0 . 0004 , FcγRIV P = 0 . 0284 ) ( D ) Phosphorylated Syk ( P - Syk ) and total Syk ( T - Syk ) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone , together with antibody 2D8 ( Aβ - 2D8 ) , or an isotype control ( Aβ - IC ) . Actin was used as a loading control . ( E ) Quantification of P - Syk normalized to T - Syk . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0490 , genotype P = 0 . 0898 , treatment P < 0 . 0001 . Bonferroni - corrected pair - wise post hoc tests , * * P = 0 . 0075 vs . wt ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative histograms for Fcγ-receptors-PE (FcγR-PE) expression levels as used for quantification. Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR (I, IIB/III and IV).(B) Relative quantification of cell surface levels of FcγR molecules. Absolute number of cell surface FcγR-PE molecules was determined by the BD QuantiBRITE® method (see methods section for details) and normalized to expression levels of the respective wt control. (n=4, +/- SEM, t test, two-tailed; wt vs. ko: FcγRI-PE P=0.0008, FcγRIIB/III-PE P=0.0033, FcγRIV-PE P=0.7001).(C) mRNA levels of FcγR are increased in Trem2 ko BMDM. Fold changes of the respective FcγR (I, IIB, III, IV) mRNA levels in Trem2 ko BMDM were determined by quantitative real-time PCR. (n=6, +/- SEM; one-sample t test, two-tailed; wt vs. ko: FcγRI P=0.0118, FcγRIIB P=0.0006, FcγRIII P=0.0004, FcγRIV P=0.0284)(D) Phosphorylated Syk (P-Syk) and total Syk (T-Syk) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone, together with antibody 2D8 (Aβ-2D8), or an isotype control (Aβ-IC). Actin was used as a loading control.(E) Quantification of P-Syk normalized to T-Syk. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0490, genotype P=0.0898, treatment P<0.0001. Bonferroni-corrected pair-wise post hoc tests, ** P =0.0075 vs. wt).


Figure 3 ( B ) mAb11 but not the IC co - localized with methoxy - X04 . BMDM ( C ) or primary microglia ( D ) were cultured on cryosections pre - incubated with mAb11 ( 1 µg / ml ) . After 24 h , sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy - X04 staining to visualize Aβ . Note that both cell types internalize Aβ into intracellular vesicles ( right panels show enlargement of insets ) . Scale bar : 10µm .

O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) mAb11 but not the IC co-localized with methoxy-X04.BMDM (C) or primary microglia (D) were cultured on cryosections pre-incubated with mAb11 (1 µg/ml). After 24 h, sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy-X04 staining to visualize Aβ. Note that both cell types internalize Aβ into intracellular vesicles (right panels show enlargement of insets). Scale bar: 10µm.