Journal: Stem Cell Research & Therapy

Article Title: BACH1 recruits STAT3 to enhance leukemia inhibitory factor receptor activity and augments the self-renewal capacity of mouse embryonic stem cells

doi: 10.1186/s13287-025-04578-x

Figure Lengend Snippet: Higher expression levels of Bach1 and Lifr in ICM cells. ( A ) Representative snapshots of ChIP-seq tracks and motif analysis show BACH1 binding on the enhancer of Lifr . ( B ) t-Distributed stochastic neighbor embedding (tSNE) analysis of 133 mouse blastocyst cells. ( C ) Violin plots depict the normalized expression of Bach1 and Lifr and the pluripotent genes, including Nanog , Oct4 , Sox2 , Esrrb , and TE marker genes, such as Gata3 and Cdx2 . ( D ) RT-qPCR analysis of mesendodermal and neuroectodermal genes expression in WT and Bach1 -KO mESCs at Day 0, 2 or 4 of EB differentiation following withdrawal of mLIF. Data are presented as mean ± SD, and statistical analysis was performed by the t-test. ( n = 3 per group). ( E ) RT-qPCR analysis of mesendodermal and neuroectodermal genes expression in WT and Dox Bach1 mESCs at Day 0, 2 or 4 of EB differentiation following withdrawal of mLIF. Data are presented as mean ± SD, and statistical analysis was performed by the t-test. ( n = 3 per group). ( F ) RT-qPCR analysis of pluripotency-associated genes expression in WT and Bach1 -KO mESCs at Day 0, 2 or 4 of EB differentiation following withdrawal of mLIF. Data are presented as mean ± SD, and statistical analysis was performed by one-way ANOVA followed by the Tukey test. ( n = 3 per group). ( G ) RT-qPCR analysis of pluripotency-associated genes expression in WT and Dox Bach1 mESCs at Day 0, 2 or 4 of EB differentiation following withdrawal of mLIF. Data are presented as mean ± SD, and statistical analysis was performed by one-way ANOVA followed by the Tukey test. ( n = 3 per group)

Article Snippet: Antibodies to Phospho-STAT3 (#AP0705, ABclonal, Wuhan, China), STAT3 (#12640S, Cell Signaling Technology, Danvers, MA, USA), SOX2 (#23064S, Cell Signaling Technology, Danvers, MA, USA), OCT-4 A (#83932S, Cell Signaling Technology, Danvers, MA, USA), Phospho-JAK2-Y1007/1008 (#AP0531, ABclonal, Wuhan, China), JAK2 (#A19629, ABclonal, Wuhan, China), BACH1 (#sc-271211, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-ACTIN (#20536-1-AP, Proteintech, Rosemont, IL, USA), LIFR (#sc-515337, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Histone 3 (#4620S, Cell Signaling Technology, Danvers, MA, USA), β-Tubulin (# T0023, Affinity, Jiangsu, China) were used.

Techniques: Expressing, ChIP-sequencing, Binding Assay, Marker, Quantitative RT-PCR

Journal: Stem Cell Research & Therapy

Article Title: BACH1 recruits STAT3 to enhance leukemia inhibitory factor receptor activity and augments the self-renewal capacity of mouse embryonic stem cells

doi: 10.1186/s13287-025-04578-x

Figure Lengend Snippet: Loss of Bach1 diminishes the expression of Lifr in mESCs. ( A ) Heatmap illustrating RNA expression in WT and Bach1 -KO mESCs from RNA-Seq analysis. ( B ) KEGG and GO analysis of decreased genes to identify the most significantly regulated biological processes in Bach1 -KO mESCs compared to WT mESCs based on the RNA-seq data. ( C ) Gene Set Enrichment Analysis (GSEA) of differentially expressed genes between Bach1 -KO mESCs and WT mESCs, focusing on genes associated with response to leukemia inhibitory factor (NES = 1.22, FDR = 0.17). ( D ) Gene Set Enrichment Analysis (GSEA) of differentially expressed genes between Bach1 -KO mESCs and WT mESCs, focusing on genes associated with embryonic stem cell pluripotency (NES = 1.22, FDR = 0.12). ( E-F ) The mRNA levels of Lifr were measured in WT or Bach1 -KO mESCs ( E ), and in -Dox or Dox Bach1 mESCs ( F ). Data are presented as mean ± SD, and statistical analysis was performed by the t-test. ( n = 3 per group)

Article Snippet: Antibodies to Phospho-STAT3 (#AP0705, ABclonal, Wuhan, China), STAT3 (#12640S, Cell Signaling Technology, Danvers, MA, USA), SOX2 (#23064S, Cell Signaling Technology, Danvers, MA, USA), OCT-4 A (#83932S, Cell Signaling Technology, Danvers, MA, USA), Phospho-JAK2-Y1007/1008 (#AP0531, ABclonal, Wuhan, China), JAK2 (#A19629, ABclonal, Wuhan, China), BACH1 (#sc-271211, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-ACTIN (#20536-1-AP, Proteintech, Rosemont, IL, USA), LIFR (#sc-515337, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Histone 3 (#4620S, Cell Signaling Technology, Danvers, MA, USA), β-Tubulin (# T0023, Affinity, Jiangsu, China) were used.

Techniques: Expressing, RNA Expression, RNA Sequencing

Journal: Stem Cell Research & Therapy

Article Title: BACH1 recruits STAT3 to enhance leukemia inhibitory factor receptor activity and augments the self-renewal capacity of mouse embryonic stem cells

doi: 10.1186/s13287-025-04578-x

Figure Lengend Snippet: BACH1 promotes the enhancer activity of Lifr and activates LIFR-STAT3 signaling in mESCs. ( A ) ChIP-qPCR assays confirmed BACH1 occupancy at multiple regions of the Lifr gene enhancer, using IgG as a negative control. Data are presented as mean ± SD, and statistical analysis was performed by the t-test. ( n = 3 per group). ( B ) The luciferase reporter assay. WT or mutant Lifr luciferase reporter was transfected into -Dox or Dox Bach1 mESCs, and luciferase activity was evaluated 24 h post-transfection. Data are presented as mean ± SD, and statistical analysis was performed by the t-test. ( n = 6 per group). ( C ) Western blot analysis was conducted to assess the phosphorylation and total protein levels of JAK2 and STAT3, as well as the protein levels of LIFR in WT mESCs and Bach1 -KO mESCs. ( D ) Quantitative analysis of protein levels in ( C ), normalized to the control group (WT), based on data from three independent experiments. Data are presented as mean ± SD, and statistical analysis was performed by the t-test. Full-length blots are presented in Supplementary Fig. . ( n = 3 per group). ( E ) Western blot analysis was conducted to assess the phosphorylation and total protein levels of STAT3, as well as the protein levels of LIFR in WT mESCs and Bach1 -KO mESCs with varying concentrations of mLIF (0 U, 50 U, 200 U, 500 U, and 1000 U) treated for 48 h. Quantitative analysis was in Fig. 3F and Supplementary Fig. . Full-length blots are presented in Supplementary Fig. . ( n = 3 per group). ( F ) Quantitative analysis of P-STAT3/STAT3 protein levels in ( E ). Data are presented as mean ± SD, and statistical analysis was performed by one-way ANOVA followed by the Tukey test

Article Snippet: Antibodies to Phospho-STAT3 (#AP0705, ABclonal, Wuhan, China), STAT3 (#12640S, Cell Signaling Technology, Danvers, MA, USA), SOX2 (#23064S, Cell Signaling Technology, Danvers, MA, USA), OCT-4 A (#83932S, Cell Signaling Technology, Danvers, MA, USA), Phospho-JAK2-Y1007/1008 (#AP0531, ABclonal, Wuhan, China), JAK2 (#A19629, ABclonal, Wuhan, China), BACH1 (#sc-271211, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-ACTIN (#20536-1-AP, Proteintech, Rosemont, IL, USA), LIFR (#sc-515337, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Histone 3 (#4620S, Cell Signaling Technology, Danvers, MA, USA), β-Tubulin (# T0023, Affinity, Jiangsu, China) were used.

Techniques: Activity Assay, ChIP-qPCR, Negative Control, Luciferase, Reporter Assay, Mutagenesis, Transfection, Western Blot, Phospho-proteomics, Control

Journal: Stem Cell Research & Therapy

Article Title: BACH1 recruits STAT3 to enhance leukemia inhibitory factor receptor activity and augments the self-renewal capacity of mouse embryonic stem cells

doi: 10.1186/s13287-025-04578-x

Figure Lengend Snippet: Colocalization of BACH1 and STAT3 at gene enhancers in mESCs. ( A ) Heatmaps depict the genome-wide ChIP-seq binding profiles of BACH1, STAT3, H3K4me3, H3K27ac, H3K4me1, and H3K27me3 at BACH1 enriched regions (region TSS ± 10 kb). ( B ) A Venn diagram illustrates the genes enriched by BACH1, STAT3, H3K4me1, and H3K27ac. ( C ) The known transcription factors’ motifs at BACH1 enriched regions. ( D ) Representative snapshots of ChIP-seq tracks showcase BACH1 and STAT3 binding on the enhancer of Lifr , Oct4 , and Socs3 . ( E ) Co-immunoprecipitation of BACH1 and STAT3 in mESCs. BACH1 was immunoprecipitated from mESCs, and the presence of STAT3 in the precipitate was assessed via Western blot. Full-length blots are presented in Supplementary Fig. . ( F ) Bacterially expressed His-tagged STAT3 was incubated with GST-tagged BACH1 protein (BACH1-GST). Subsequently, the reaction products were precipitated using glutathione-Sepharose 4B beads, and STAT3 was detected in the precipitate by Western blot analysis using anti-His antibodies. Full-length blots are presented in Supplementary Fig. . ( G ) The STAT3 binding to enhancers of Sox2 , Oct4 , and Lifr was assessed in WT mESCs or Bach1 -KO mESCs using ChIP-qPCR. Data are presented as mean ± SD. ( n = 3 per group). ( H ) mESCs were treated with varying concentrations of mLIF. Then, western blot analysis was conducted to assess the phosphorylation of STAT3. Full-length blots are presented in Supplementary Fig. . The ChIP assay was performed after western blot analysis confirmed that P-STAT3 levels were comparable between WT and Bach1 -KO mESCs. The binding of STAT3 to the enhancer regions of Sox2 , Oct4 , Lifr , and Myc was evaluated in WT mESCs or Bach1 -KO mESCs using ChIP-qPCR. Data are presented as mean ± SD ( n = 3 per group)

Article Snippet: Antibodies to Phospho-STAT3 (#AP0705, ABclonal, Wuhan, China), STAT3 (#12640S, Cell Signaling Technology, Danvers, MA, USA), SOX2 (#23064S, Cell Signaling Technology, Danvers, MA, USA), OCT-4 A (#83932S, Cell Signaling Technology, Danvers, MA, USA), Phospho-JAK2-Y1007/1008 (#AP0531, ABclonal, Wuhan, China), JAK2 (#A19629, ABclonal, Wuhan, China), BACH1 (#sc-271211, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-ACTIN (#20536-1-AP, Proteintech, Rosemont, IL, USA), LIFR (#sc-515337, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Histone 3 (#4620S, Cell Signaling Technology, Danvers, MA, USA), β-Tubulin (# T0023, Affinity, Jiangsu, China) were used.

Techniques: Genome Wide, ChIP-sequencing, Binding Assay, Immunoprecipitation, Western Blot, Incubation, ChIP-qPCR, Phospho-proteomics

Journal: Stem Cell Research & Therapy

Article Title: BACH1 recruits STAT3 to enhance leukemia inhibitory factor receptor activity and augments the self-renewal capacity of mouse embryonic stem cells

doi: 10.1186/s13287-025-04578-x

Figure Lengend Snippet: BACH1 maintains the self-renewal capability of mESCs through LIFR. ( A ) Luciferase reporter assay. The WT Lifr luciferase reporter plasmid was transfected into mESCs with or without Dox-inducible Bach1 overexpression for 24 h, followed by Stat3 knockdown via siRNA for 48 h. Luciferase activity was evaluated 24 h post-transfection. The analysis was based on six independent experiments, and the data are expressed as mean ± SD. Statistical significance was determined using 2-way ANOVA followed by the Tukey test ( n = 6 per group). ( B ) Western blot analysis was performed to evaluate the protein levels of LIFR, SOX2, OCT4, and STAT3 in mESCs. The experiments were conducted under conditions with or without Bach1 overexpression for 24 h, followed by Stat3 knockdown via siRNA for 48 h. Full-length blots are provided in Supplementary Fig. . ( C ) Quantitative analysis of the protein levels shown in ( B ) was performed, with data normalized to the control group. The analysis was based on three independent experiments, and the data are expressed as mean ± SD. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test ( n = 3 per group). ( D ) Alkaline phosphatase (AP) staining of mESCs colonies involved the withdrawal of mLIF for three days, followed by the knockdown of Lifr via siRNA for 48 h after overexpressing Bach1 through a 24-hour treatment with Dox. ( E ) Quantitative analysis assessing the proportions of total cell clones categorized as undifferentiated, partially differentiated, and fully differentiated cells. This analysis was performed in both WT and Bach1 overexpression mESCs, with or without Lifr knockdown via siRNA. Statistical analysis was performed by 2-way ANOVA followed by the Tukey test ( n = 4 per group). ( F ) Western blot analysis was conducted to assess the phosphorylation and total protein levels of JAK2 and STAT3, as well as the protein levels of LIFR, SOX2, and OCT4 in mESCs. This was performed with or without Bach1 overexpression for 24 h, followed by Lifr knockdown via siRNA for 48 h. Full-length blots are presented in Supplementary Fig. . ( G ) Quantitative analysis of the levels of protein in ( F ), normalized to the control group, was carried out based on data from three independent experiments. The data are presented as mean ± SD, and statistical analysis was performed by 2-way ANOVA followed by the Tukey test ( n = 3 per group)

Article Snippet: Antibodies to Phospho-STAT3 (#AP0705, ABclonal, Wuhan, China), STAT3 (#12640S, Cell Signaling Technology, Danvers, MA, USA), SOX2 (#23064S, Cell Signaling Technology, Danvers, MA, USA), OCT-4 A (#83932S, Cell Signaling Technology, Danvers, MA, USA), Phospho-JAK2-Y1007/1008 (#AP0531, ABclonal, Wuhan, China), JAK2 (#A19629, ABclonal, Wuhan, China), BACH1 (#sc-271211, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-ACTIN (#20536-1-AP, Proteintech, Rosemont, IL, USA), LIFR (#sc-515337, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Histone 3 (#4620S, Cell Signaling Technology, Danvers, MA, USA), β-Tubulin (# T0023, Affinity, Jiangsu, China) were used.

Techniques: Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Over Expression, Knockdown, Activity Assay, Western Blot, Control, Staining, Clone Assay, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

doi: 10.1074/jbc.m700254200

Figure Lengend Snippet: FIGURE 1. Soluble, recombinant Bach1 isolated from E. coli. BL21(DE3) cells, transformed with an expression plasmid encoding the 100-kDa recom- binant, His-tagged Bach1 protein (22), were grown in Luria Bertani medium containing 1 M sorbitol and 250 M betaine (31) and induced with 1 mM iso- propyl-1-thio--D-galactopyranoside for 12 h at room temperature. Washed cells were sonicated and centrifuged to remove debris, and Bach1 protein was isolated from the supernatant solution by Ni-NTA affinity chromatogra- phy. Analysis of pooled fractions of the soluble, recombinant protein used SDS gel electrophoresis and staining with Coomassie Blue or Western blot- ting with horseradish peroxidase-conjugated anti-His tag, mouse mono- clonal antibody detected by chemiluminescence (ECL). CB, Coomassie Blue; WB, Western blot.

Article Snippet: The Bach1 overexpression plasmid was obtained from Origene (catalogue number SC107917); cells were transfectedwith 1 g of the plasmid DNA 24 h after seeding, using FuGENE 6 transfection reagent (Roche Applied Science).

Techniques: Recombinant, Isolation, Transformation Assay, Expressing, Plasmid Preparation, Sonication, SDS-Gel, Electrophoresis, Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

doi: 10.1074/jbc.m700254200

Figure Lengend Snippet: FIGURE 2. Bach1 binds to FTL and FTH MARE/ARE DNA in chromatin of cultured cells. Bach1 in chromatin from Hepa1c1c7 cells was analyzed by immunoprecipitation with Bach1 antibodies or normal rabbit serum (NRS). Gel images are shown of PCR products of fth (ferritin H) and ftl (ferritin L) with -G (-globin HS-2) and ho1 (HO1 E1) as positive controls and Mcm5 pro- moter as a negative control. The results are representative of three independ- ent experiments.

Article Snippet: The Bach1 overexpression plasmid was obtained from Origene (catalogue number SC107917); cells were transfectedwith 1 g of the plasmid DNA 24 h after seeding, using FuGENE 6 transfection reagent (Roche Applied Science).

Techniques: Cell Culture, Immunoprecipitation, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

doi: 10.1074/jbc.m700254200

Figure Lengend Snippet: FIGURE 3. Increasing Bach1 expression increases repression of MARE/ ARE genes. HepG2 cells were transfected with DNA encoding Bach1 in plas- mid in pCMV6-XL6 (Origene) (1 g DNA/well). Control cells were transfected with pCMV6-XL6. Me2SO (0.2%), with or without hemin (80 M) was added 20 h after transfection; after 24 h RNA concentrations of Bach1, ftl, qr, trr, and ho1 were determined by real-time PCR. The data were normalized to the control values shown as the dashed line. The experiments were carried out at the same time for both promoters, allowing the use of the same control within each of three independent experiments. Data are presented as aver- ages (n 3) S.D. *, significantly different from control (p 0.05).

Article Snippet: The Bach1 overexpression plasmid was obtained from Origene (catalogue number SC107917); cells were transfectedwith 1 g of the plasmid DNA 24 h after seeding, using FuGENE 6 transfection reagent (Roche Applied Science).

Techniques: Expressing, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

doi: 10.1074/jbc.m700254200

Figure Lengend Snippet: FIGURE 4. Bach1 binding to DNA in vitro is selective for the MARE/ARE sequences and abrogated by heme. Left, effect of MARE/ARE sequence on competitive EMSA. Double-stranded, [32P]FTL DNA (40 base pairs) in 60 mM KCl, 24 mM HepesNa, 4 mM MgCl2, 5% glycerol, and 2% 2-mercaptoethanol, pH 7.2) was incubated with Bach1 (protein/DNA molar ratio 72:1; DNA 1.6 pM) with or without 100 excess, cold wild-type ftl probe (lane 3) or mutated ftl (TGAC/GACA) probe (lane 4). Right, effect of cobalt protoporphyrin on MARE/ARE interactions. Wild-type [32P]ftl MARE/ARE was mixed with recom- binant Bach1 at a protein/DNA ratio of 36:1 with or without 40 M metallo- porphyrin (lane 3, or cobalt protoporphyrin IX, lane 4); the metalloporphyrins were dissolved in Me2SO, with a final Me2SO concentration of 0.2% in all samples, including control. The data are representative of three independent experiments. Arrows show the origin of each gel.

Article Snippet: The Bach1 overexpression plasmid was obtained from Origene (catalogue number SC107917); cells were transfectedwith 1 g of the plasmid DNA 24 h after seeding, using FuGENE 6 transfection reagent (Roche Applied Science).

Techniques: Binding Assay, In Vitro, Sequencing, Incubation, Concentration Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

doi: 10.1074/jbc.m700254200

Figure Lengend Snippet: FIGURE 5. Bach1 protein concentrations required for full DNA binding varyamongdifferentMARE/AREsequences.Double-stranded,[32P]MARE/ AREDNAsequences(40basepairs)fromsixdifferentMARE/AREgenes,ftl,fth, trr, qr, ho1, and -globin, in 20 l of 60 mM KCl, 24 mM HepesNa, 4 mM MgCl2, 5% glycerol, and 2% 2-mercaptoethanol, pH 7.2, were incubated on ice for 15 min with increasing amounts of recombinant Bach1 protein. Protein: [32P]DNA ratios were lane 1, 0; lane 2, 9:1; lane 3, 18:1; lane 4, 36:1; lane 5, 72:1; the concentration of DNA was 80 nM. Free DNA and bound DNA were resolved by electrophoresis (nondenaturing, 5% acrylamide gels run at 4 °C); dried gels were analyzed by phosphoimagery and quantified with Image- quant. Arrows show the origin of each gel. Three groups of DNAprotein com- plex with significantly different stabilities were observed (See Table 1 for Kd values): (i) stability -globin (ftl and fth);(ii) stability significantly different from -globin (ho1, qr, trr); and (iii) significantly (p 0.05) different from trr (-globin, fth, ftl, ho1, and qr).

Article Snippet: The Bach1 overexpression plasmid was obtained from Origene (catalogue number SC107917); cells were transfectedwith 1 g of the plasmid DNA 24 h after seeding, using FuGENE 6 transfection reagent (Roche Applied Science).

Techniques: Binding Assay, Incubation, Recombinant, Concentration Assay, Electrophoresis

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1/BCLAF1 Forms an mRNA Splicing Complex which Is Recruited to Target Gene Promoters and Transcripts following DNA Damage (A) Coimmunoprecipitation assays demonstrating that BCLAF1 interacts with the spliceosome proteins Prp8, U2AF65, U2AF35, and SF3B1 in both the presence and absence of DNA damage. (B) Coimmunoprecipitation assays demonstrating DNA damage-induced interaction between BRCA1 and the spliceosome proteins Prp8, U2AF65 U2AF35, and SF3B1 in response to DNA damage. Additionally, depletion of BCLAF1 results in abrogation of DNA damage-induced interaction between BRCA1 and these proteins. (C) BRCA1, BCLAF1, and U2AF65 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP , BACH1 , and EXO1 promoters irrespective of DNA damage in control (siCtrl) cells. The ChIPs also demonstrate that BCLAF1 and U2AF65 are recruited to these promoters only in etoposide-treated cells and that depletion of BRCA1 or BCLAF1 results in loss of DNA damage-induced BCLAF1 and U2AF65 recruitment, respectively. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM. (D) BRCA1, BCLAF1, and U2AF65 RIP-qRT-PCRs demonstrating that BRCA1, BCLAF1, and U2AF65 only bind to ATRIP , BACH1 , and EXO1 mRNAs in response to DNA damage. In addition, depletion of BCLAF1 results in loss of BRCA1 and U2AF65 mRNA binding to all three transcripts. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM. See also Figure S3 .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Binding Assay, Control

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: The BRCA1/BCLAF mRNA Splicing Complex Promotes the Splicing and Stability of ATRIP , BACH1 , and EXO1 Transcripts following DNA Damage (A) Ratio of postspliced to prespliced ATRIP , BACH1 , and EXO1 mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells mock treated or treated with etoposide. mRNA levels were assessed by qRT-PCR using exon 9-exon 10 (post-spliced- ATRIP ) and exon 9-intron 9 (pre-spliced- ATRIP ), exon 15-exon 16 (post-spliced- BACH1 ) and exon 15-intron 15 (pre-spliced- BACH1 ), and exon 1-exon 2 (post-spliced- EXO1 ) and exon 1-intron 1 (pre-spliced- EXO1 ) primers and normalized to ACTB mRNA. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM. Significance of changes in splicing ratios was assessed using Student’s two-tailed t test with significant changes indicated by ∗∗ p < 0.01. (B) Semiquantitative PCR analysis of a cDNA generated from DNase-treated RNA, collected from control (siCtrl) and BRCA1- or BCLAF1-depleted cells, mock treated or treated with Etoposide. Primers targeting two independent intronic regions within ATRIP , BACH1 , and EXO1 and a single intronic region within ATM and CHEK2 (control genes) were used for semiquantitative PCR analysis. Exon-spanning primers targeted against ACTB were used as a loading control. (C) Normalized expression of prespliced and postspliced mRNAs evaluated in (A). (D) Expression levels of postspliced and prespliced ATRIP mRNAs in control (siCtrl) and BRCA1- or BCLAF1-depleted cells, transfected with control siRNA (siCtrl) or depleted of SMG1 (siSMG1), a key regulator of the non-sense-mediated decay pathway. Normalized expression levels were quantified as in (A). Graphs represent the mean normalized expression from three independent experiments ± SEM. See also and .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Control, Quantitative RT-PCR, Two Tailed Test, Generated, Expressing, Transfection

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1 Ser-1423 Phosphorylation Is Required for BCLAF1 Recruitment and Target Gene Splicing following DNA Damage (A) BRCA1, pS1423-BRCA1, and BCLAF1 ChIP-qPCRs demonstrating constitutive binding of BRCA1 to ATRIP , BACH1 , and EXO1 promoters irrespective of DNA damage and DNA damage-dependent enrichment of pS1423-BRCA1 and BCLAF1 on these promoters. Graphs represent the mean fold enrichment quantified from three independent experiments ± SEM. (B–D) Ratio of postspliced to prespliced ATRIP , BACH1 , and EXO1 mRNA in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt-BRCA1), or S1423A Flag-BRCA1 (S1423A-BRCA1). Cells were mock treated or treated with etoposide. mRNA levels were assessed as described in Figure 4 A. Graphs represent the mean ratios of postspliced/prespliced mRNA from three independent experiments ± SEM ATRIP , BACH1 , and EXO1 splicing is upregulated in cells expressing wild-type BRCA1 but not S1423A-BRCA1, indicating that phosphorylation of BRCA1 S1423 is required for DNA damage-induced splicing. (E–G) FLAG and BCLAF1 ChIP-qRT-PCR analysis carried out in BRCA1-deficient cells (HCC1937) transfected with empty vector (EV), wild-type Flag-BRCA1 (wt), or S1423A Flag-BRCA1 (S1423A). Cells were mock treated or treated with etoposide. Graphs represent the mean fold enrichment from three independent experiments ± SEM. (H) Representative western blots demonstrating wild-type and S1423A Flag-BRCA1 in HCC1937 cells used for splicing assays and ChIPs presented above. See also Figure S6 .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Phospho-proteomics, Binding Assay, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1/BCLAF1-Mediated mRNA Splicing Is Required for Maintenance of ATRIP, BACH1, and EXO1 Protein Expression (A) Representative western blots demonstrating that depletion of either BRCA1 or BCLAF1 results in downregulated expression of ATRIP, BACH1, and EXO1 proteins in response to DNA damage. (B) Representative western blot demonstrating DNA damage-dependent accumulation of ATRIP, BACH1, and EXO1 proteins over time following inhibition of proteosomal mediated protein degradation with MG132 (10 μM). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to MG132 treatment. (C) Representative western blots demonstrating DNA damage-dependent increased protein turnover in control and BRCA1- or BCLAF1-depleted cells following inhibition of protein translation with Cyclohexamide (10 μg/mL). Cells were mock treated or treated with etoposide (1 μM) for 30 min prior to Cyclohexamide treatment. (D) Representative western blots demonstrating BRCA1 and BCLAF depletion in cells used for experiments shown in (C). (E–G) Quantification of ATRIP, BACH1, and EXO1 protein levels shown in (C). Image densitometry values were normalized to 0 hr and decay curves fitted and used to calculate protein half-lives.

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Expressing, Western Blot, Inhibition, Control

Journal: Molecular Cell

Article Title: Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

doi: 10.1016/j.molcel.2014.03.021

Figure Lengend Snippet: BRCA1/BCLAF1-Mediated mRNA Splicing of ATRIP, BACH1, and EXO1 Promotes Resistance to DNA Damage and Efficient DNA Repair (A) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in BRCA1- or BCLAF1-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. (B) Clonogenic survival assays demonstrating that depletion of BCLAF1 or U2AF65 induces sensitivity to IR in 293T cells. Mean surviving fraction of three independent experiments is plotted ± SEM. (C) Representative immunofluorescent staining of γ-H2AX marked DNA damage in untreated 293T cells depleted of either BCLAF1 or U2AF65 and 1 and 24 hr following 2Gy IR. (D. Quantification of three independent experiments described above (≥200 cells were scored/experiment). The mean fraction of cells containing ≥5 γ-H2AX foci is plotted ± SEM. Significant differences in the fraction of cells containing ≥5 γ-H2AX foci were assessed using Student’s two-tailed t test and are indicated by ∗∗∗ p < 0.001. (E) Clonogenic survival assays demonstrating that ectopic expression of ATRIP, BACH1, and EXO1 (A/B/E) in U2AF65-depleted 293T cells partially rescues their sensitivity to IR. Mean surviving fraction of three independent experiments is plotted ± SEM. See also Figure S7 .

Article Snippet: Myc-DDK tagged ATRIP (RC223562), BACH1 (RC224085), and EXO1 (RC200547) plasmids were purchased from Origene.

Techniques: Expressing, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE142102 ( n = 226) dataset of TNBC patients (Pearson correlation coefficient r = 0.3245, P < 0.0001). ( B ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE103091 ( n = 238) dataset of TNBC patients (Pearson correlation coefficient r = 0.2120, P < 0.001). ( C ) Western blot showing SDCBP, BACH1, and HO-1 protein expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells. ( D ) The expression levels of SDCBP and BACH1 protein in Fig. EV1C were quantified using densitometry and normalized to the housekeeping protein α-tubulin ( n = 3). ( E ) Real-time qPCR showing SDCBP and BACH1 mRNA expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells ( n = 3). Quantitative data were normalized to β-actin expression. ( F ) Western blot showing SDCBP and HO-1 protein expression in MDA-MB-231 cells transfected with scramble or BACH1 siRNA. ( G ) Left, western blot showing the protein expression of SDCBP in the scramble and in several SDCBP-KO MDA-MB-231 subclones generated using CRISPR-Cas9 system; Right, real-time qPCR showing the SDCBP mRNA expression in scramble and in SDCBP-KO MDA-MB-231 subclones ( n = 3). ( H ) Real-time qPCR showing the mRNA expression of BACH1 in MDA-MB-231 cells, in scramble and in SDCBP-KO MDA-MB-231 subclone#2 and subclone#12 ( n = 3). ( I ) Immunofluorescence staining was used to visualize SDCBP (green color) and BACH1 (red color) in scramble and in SDCBP-KO MDA-MB-231 cells. DAPI (blue color) was used to stain the nucleus ( n = 3); Representative confocal immunofluorescence images are shown. Scale bar = 20 µm. ( J ) Western blot showing BACH1 and HO-1 protein expression in 4T1 cells infected with scramble or adenoviral SDCBP shRNA. ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1, NQO1 , and GLCL ) in 4T1 cells transfected with scramble or SDCBP siRNA ( n = 3); mRNA expression of KEAP1 was the negative control. Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( D , E , G , H ) or two-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Expressing, Western Blot, Transfection, Generated, CRISPR, Immunofluorescence, Staining, Infection, shRNA, Negative Control

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunohistochemistry staining against the SDCBP and BACH1 protein in human TNBC-derived tissue microarray sections ( n = 78). Representative images showing the co-expression of SDCBP and BACH1 in the same section. Normal breast cancer tissues were considered as the negative control. Scale bar = 20 µm. ( B ) Pearson correlation coefficient (r = 0.5772, P < 0.0001) between SDCBP and BACH1 expression in ( A ). ( C ) Western blot showing BACH1 and HO-1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( D ) Real-time qPCR showing BACH1 mRNA expression in Hs578T cells transfected with a control vector or a Myc-SDCBP-expressing vector ( n = 3). ( E ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1 , and CXCR4 ) in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( F ) Western blot showing BACH1 protein expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA. ( G ) Real-time qPCR showing BACH1 mRNA expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA ( n = 3). ( H ) Left, Representative images of immunofluorescence staining to visualize SDCBP ( green color ) and BACH1 ( red color ) expression in MDA-MB-231 cells transfected with a scramble siRNA or SDCBP siRNA. DAPI ( blue color) was used to stain the nucleus ( n = 3); Scale bar = 50 µm. Right, fluorescence levels of SDCBP and BACH1 were quantified based on their spectral densities. ( I ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1, MMP13 , and CXCR4 ) in MDA-MB-231 cells transfected with scramble siRNA or SDCBP siRNA ( n = 3). ( J ) Western blot showing SDCBP and BACH1 protein expression in scramble control and two SDCBP-KO MDA-MB-231 clones (KO#2, KO#12) generated using CRISPR-Cas9 system ( n = 3). ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated metastatic genes ( HK2 , MMP1 , CXCR4, GAPDH , and VEGF ) in scramble control and SDCBP-KO MDA-MB-231 cells. ( L ) The reconstitution of SDCBP recovers BACH1 protein expression in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1 protein expression in scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector. The arrows indicate the endogenous (Endo) and exogenous (Exo) SDCBP. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( D , E , G , I ), two-way ANOVA ( H ), or one-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Microarray, Expressing, Negative Control, Western Blot, Transfection, Control, Plasmid Preparation, Infection, shRNA, Immunofluorescence, Fluorescence, Clone Assay, Generated, CRISPR, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA. ( B ) Colony formation of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . Migration of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . ( D ) Western blot showing SDCBP and Flag-BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector. Cell proliferation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( G ) Wound closure of scratched MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( H ) Schematic of various SDCBP mutant constructs generated using the Myc-SDCBP plasmid. ( I ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with the indicted SDCBP constructs. Right, quantification of BACH1 levels using densitometry ( n = 3). ( J ) Top, schematic of the PDZ1 construct. Bottom, western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP or Myc-SDCBP_PDZ1 plasmid. ( K ) Migration of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( L ) Colony formation of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( M ) Tumor volumes from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 5 or 7 mice/group). ( N ) Tumor weights in Fig. 2M ( n = 7 mice/group); See also Fig. . ( O ) Tumor volumes from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group); See also Fig. – , . Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( B , , I , K , L , N ), two-tailed Student’s t test ( , F , O ), or two-way ANOVA ( G , M ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Migration, Mutagenesis, Construct, Generated, Injection, Control, Stable Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing the expression of SDCBP and Flag-SDCBP in Fig. . ( B ) Tumor weights in Fig. ( n = 5 mice/group). ( C ) Tumor volume in Fig. ( n = 5 mice/group). ( D ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 7 mice/group). Representative images of IHC staining are shown. Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( E ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, or SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group). Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( F ) TCGA data analysis showing association between SDCBP mRNA expression and overall survival ( n = 392) and lymph node status ( n = 98) of TNBC patients. ( G ) TCGA data analysis showing association between BACH1 mRNA expression and overall survival ( n = 2032) and lymph node status ( n = 98) of TNBC patients. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( B ) or two-tailed Student’s t test ( C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Isolation, Injection, Control, Stable Transfection, Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Representative images of colony formation in Fig. . ( B ) Representative images of the migrated cells in Fig. . Scale bar = 200 µm. ( C ) Representative images of colony formation in Fig. . ( D ) Representative images of wounding migration in Fig. . Scale bar = 200 µm. ( E ) Cell proliferation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. Cell proliferation was estimated by an automatic cell counter at the indicated time points ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The clonogenic ability was assessed and quantified based on the absorbance at 600 nm and normalized to the control ( n = 3). ( G ) Migration of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The number of migrated cells were counted and expressed as percentages ( n = 3). ( H ) Representative images of the migrated cells in Fig. . Scale bar = 500 µm. ( I ) Representative images of colony formation in Fig. . Scale bar = 1000 µm. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( E – G ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Migration, Transfection, Control, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without MG132 proteasome inhibitor. ( B ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( C ) Left, western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( D ) Western blot showing BACH1, FBXO22, and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with HA-FBXO22-expressiong vector with or without Myc-SDCBP-expressing vector. ( F ) In vitro ubiquitylation assay of the recombinant human BACH1 protein mediated by the FBXO22 complex. Active recombinant human UbcH5a protein was used as the E2 ubiquitin-conjugating enzyme for FBXO22 complex-mediated BACH1 degradative polyubiquitylation. ( G ) In vivo ubiquitylation assay showing the decrease in the K48-linked polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. ( H ) In vivo ubiquitylation assay showing the increase in the K48-linked polyubiquitylation of BACH1 by SDCBP KD in MDA-MB-231 cells transfected with the indicated plasmids. ( I ) In vitro ubiquitylation assay showing the inhibitory effect of SDCBP on the polyubiquitylation of BACH1 mediated by the FBXO22 complex. Active recombinant human protein UbcH5a and immunocomplex FBXO22 were added as described above. Recombinant human BACH1 and recombinant human SDCBP proteins were added at ratios of 1:1 (+) and 1:2 (++). Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( B , C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, In Vitro, Ubiquitin Assay, Recombinant, Ubiquitin Proteomics, In Vivo, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Left, Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( B ) Free heme level in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( C ) Free heme level in scramble and in SDCBP - KO MDA-MB-231 cells ( n = 3). ( D ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or NRF2 (encoded by NFE2L2 )-expressing vector. HO-1 protein expression was considered as the positive control. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with a control or a HO-1 (encoded by HMOX1 )-expressing vector. ( F ) Western blot showing BACH1 protein expression in Hs578T cells transfected with scramble or HO-1 siRNA. ( G ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with the HO-1 or the catalytic inactive HO-1 mutant (H25A) plasmid. ( H ) Western blot showing BACH1 and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or HOIL1 siRNA. ( I ) Western blot showing endogenous FBXO22 protein expression in several breast cancer cells. ( J ) Immunoprecipitation showing the interaction of BACH1 with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ns: none specific. ( K ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in HEK293 cells transfected with the indicated plasmids. ( L ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in MDA-MB-231 cells transfected with the indicated plasmids. ( M ) In vivo ubiquitylation assay showing the decrease in FBXO22-mediated polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( A – C ). All experiments were repeated at least three times unless otherwise indicated. P values less than 0.05 were considered statistically significant.

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Mutagenesis, Immunoprecipitation, In Vivo, Ubiquitin Assay, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunoprecipitation showing the interaction of FBXO22 with SDCBP in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( B ) Immunoprecipitation showing the interaction of SDCBP with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( C ) Co-immunoprecipitation showing the interaction of FBXO22 with BACH1 in HEK293 cells with or without SDCBP after the indicated transfections. ( D ) Schematic of experimental design to investigate the assembly of SCF FBXO22 –BACH1 complex via His Pull-down assay and endogenous IP assay in Fig. D– . ( E ) His-pulldown assay showing the interaction of FBXO22 with SKP1 in HEK293 cells with control vector or Myc-SDCBP-expressing vector after the indicated transfections. See also Appendix Fig. S . ( F ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in scramble and in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1, PTEN, and PD-L1 protein expression in A549 cells transfected with scramble or SDCBP siRNA. ( H ) Western blot showing BACH1 and PD-L1 protein expression in NCI-H1299 cells transfected with scramble or SDCBP siRNA. ( I ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( J ) In vivo ubiquitylation assay showing the inhibitory effect of SDCBP on SCF FBXO22 -mediated K48-linked polyubiquitylation of BACH1 in HEK293 cells transfected with the indicated plasmids.

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Immunoprecipitation, Transfection, Pull Down Assay, Control, Plasmid Preparation, Expressing, Western Blot, In Vivo, Ubiquitin Assay

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Crosslink immunoprecipitation showing an interaction of FBXO22 with SDCBP in Hs578T cells transfected with Myc-SDCBP-expressing vector or Myc-SDCBP_Δ4-expressing vector. Schematic showing the FBXO22 mutant constructs generated using the HA-FBXO22 plasmid. ( C ) Immunoprecipitation showing SDCBP interactions with FBXO22 and its mutant constructs in HEK293 cells transfected with the indicated plasmids. ns indicates non-specific bands. See also Fig. , . ( D ) His-Pulldown assay showing the effect of SDCBP on SKP1-CUL1-FBXO22 complex formation after the indicated transfections in HEK293 cells. See also Appendix Fig. S . ( E ) His-Pulldown assay showing the effect of SDCBP KO on the SCF FBXO22 –BACH1 complex formation in the scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or His-SKP1-expressing vector. See also Appendix Fig. S . ( F ) Immunoprecipitation showing the effect of SDCBP KD on the SCF FBXO22 –BACH1 complex formation in MDA-MB-231 cells transfected with scramble or SDCBP siRNA. ( G ) Immunoprecipitation showing the effect of SDCBP overexpression on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with control vector or Myc-SDCBP-overexpressing vector. ( H ) Immunoprecipitation showing the effect of SDCBP PDZ1 domain on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with a control vector or a Myc-SDCBP-PDZ1-overexpressing vector. .

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Generated, Control, Over Expression

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Real-time qPCR showing the mRNA expression of BACH1-regulated ETC genes ( NDUFA4 , NDUFA4L2 , NDUFC2 , and COX6B2 ) in scramble and SDCBP-KO MDA-MB-231 cells ( n = 3); See also Appendix Fig. S , . Real-time qPCR showing the mRNA expression of NDUFA4 and COX6B2 in scramble, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells transfected with SDCBP ( n = 3). ( C ) Western blots showing the expression of mitochondrial proteins NDUFA4 and COX6B2 in MDA-MB-231 cells transfected with SDCBP siRNA in the presence or absence of FLAG-BACH1-expressing vector. ( D ) ChIP-qPCR showing BACH1 enrichments in the promoter regions of NDUFA4 and COX6B2 in the scramble and SDCBP-KO MDA-MB-231 cells. Quantitative data were normalized to IgG binding expression ( n = 3); See also Appendix Fig. S . ( E ) Left, flow cytometry histogram showing the mitochondrial membrane potentials using TMRE (tetramethylrhodamine ethyl ester) staining in MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after the indicated treatments. FCCP (trifluoromethoxy carbonylcyanide phenylhydrazone). Right, quantification of TMRE fluorescence intensity ( n = 3). ( F ) Left, immunofluorescence staining and confocal imaging of the fluorescent signals for TMRE (orange-red color) in the scramble control and SDCBP-KO MDA-MB-231 cells after incubation with TMRE. DAPI (blue color) was used to stain the nucleus ( n = 7); Representative confocal images are shown; scale bars = 20 µm and 5 µm. Right, fluorescent levels of the TMRE were quantified based on their spectral densities. ( G ) Representative images of immunohistochemistry staining against the NDUFA4, BACH1, and SDCBP proteins showing a negative correlation between the expression of NDUFA4 and SDCBP in the same sections of TNBC tumor tissues. Scale bar = 20 µm. ( H ) Pearson correlation coefficients between SDCBP and NDUFA4 protein expression ( n = 64), and between BACH1 and NDUFA4 protein expression ( n = 60) in Fig. 5G. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A , D , F ), two-tailed Student’s t test , or one-way ANOVA ( E ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, ChIP-qPCR, Binding Assay, Flow Cytometry, Membrane, Staining, Fluorescence, Immunofluorescence, Imaging, Control, Incubation, Immunohistochemistry, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Cell proliferation of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with metformin for the indicated periods of time ( n = 3). ( B ) Cell viability of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin for 96 h ( n = 5). ( C ) Colony formation for MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin. Colony numbers were counted and converted to percentages by normalizing with the control groups ( n = 3). ( D – F ) Effect of the indicated treatment on 4T1 tumor growth. Tumor growth was monitored in BALB/c mice bearing 4T1 cells after mammary fat-pad injections. When the average tumor volumes reached 100 mm 3 , the mice ( n = 7 mice/group) were administered with 100 mg/kg metformin (once a day) and/or adenoviral SDCBP shRNA (1 × 10 9 PFU/mice). Black arrows indicate the day of adenoviral SDCBP shRNA injection. Final tumor volume ( E ) and weight ( F ) are shown. ( G ) Immunohistochemistry staining against SDCBP, BACH1, Ki67, NDUFA4, and COX6B2 protein in 4T1 tumors from BALB/c mice in Fig. 6A. Representative images of the IHC staining are shown. Scale bar = 50 µm for low (left) and high (right) magnification. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A – C ), one-way ANOVA ( E ), or two-tailed Student’s t test ( F ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Transfection, Control, shRNA, Injection, Immunohistochemistry, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: Schematic showing the novel oncogenic roles of SDCBP in promoting aggressiveness and mitochondrial inhibitor resistance in TNBCs. An abundance of SDCBP stabilizes the BACH1 protein by blocking E3 ubiquitin ligase SCF FBXO22 complex-targeted BACH1 for degradative ubiquitination. Mechanistically, SDCBP binds to different members of the SCF FBXO22 complex, including SKP1 and FBXO22, via its PDZ1 domain and induces SCF FBXO22 complex disassociation, suggesting that SDCBP is a key adapter regulating the activity of the E3 ubiquitin ligase SCF FBXO22 complex in the proteasomal pathway. SDCBP-induced BACH1 accumulation upregulates several pro-metastatic genes and downregulates numerous mitochondrial ETC genes, resulting in tumor progression and high resistance to metformin treatment in TNBCs. Targeting SDCBP switches the SCF FBXO22 complex to degrade BACH1 protein via the proteasome, reducing tumor aggressiveness and boosting the anti-tumor effect of metformin administration in TNBCs. .

Article Snippet: The sections were then incubated with SDCBP antibody (CAT#A5497, ABclonal, Korea), NDUFA4 antibody (Cat#PA5-51021, Invitrogen, Korea), or with BACH1 antibody (CAT#AF5776, R&D Systems, USA), and then stained using a Cell & Tissue Staining Kit (CAT#CTS005, CAT#CTS008, R&D Systems, USA), according to manufacturer’s protocol.

Techniques: Blocking Assay, Ubiquitin Proteomics, Activity Assay

Journal: Radiation Research

Article Title: Impact of RING and BRCT Domain Mutations on BRCA1 Protein Stability, Localization and Recruitment to DNA Damage

doi: 10.1667/rr1290.1

Figure Lengend Snippet: FIG. 4. BRCT domain and RING domain mutant BRCA1 proteins fail to colocalize with both BARD1 and BACH1. Panel A: Cells were stained with monoclonal antibody MS13 against BRCA1 (red) and a polyclonal antibody against BARD1 (green). Yellow signal in the merged image indicates colocalization of the two proteins. Panel B: Cells were stained with monoclonal antibody MS13 against BRCA1 (red) and a polyclonal antibody against BACH1 (green). Yellow signal in the merged image indicates colocalization of the two proteins. Nuclei are stained with DAPI in both panels. Experiments in panels A and C were performed three times with similar results. Panel C: Coimmunoprecipitation of

Article Snippet: The following primary antibodies were diluted in 0.5% nonfat dry milk/PBS-Tween-20 0.1%: BRCA1 (mouse SD118 and MS110, Calbiochem) at 1:1,000, BARD1 (rabbit BL-518, Bethyl Laboratories) at 1:5,000, BACH1 (rabbit Brip1, Novus Biologicals, Littleton, CO) at 1:5,000, cyclin D1 (rabbit monoclonal SP4, Lab Vision Corp., Fremont, CA) at 1:2,500, tubulin (mouse clone KMX-1, Chemicon) at 1:10,000. c-H2AX (mouse clone JBW301, Upstate Cell Signaling Solutions, Lake Placid, NY) was diluted in 3% BSA (Santa Cruz Biotechnology, Santa Cruz, CA)/ TBS-Tween-20 0.1% at 1:2,500.

Techniques: Mutagenesis, Staining

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: a , Total heme (WT, n = 6; PATKO, n = 8) and b , iron (n = 8) levels in BAT. c , Labile heme in mitochondrial and nuclear fractions of BAT (WT n = 5; PATKO n = 8). d , REV-ERBα and BACH1 levels in BAT. e , Genes related to heme and iron metabolism (red portions) are enriched in differentially-expressed genes. f , REV-ERBα and BACH1/2-binding motifs are enriched in genes downregulated in PATKO BAT. g , Heat map of heme and iron related genes shows a global decrease of ETC and TCA gene expression. h , UCP1 and OXPHOS proteins are reduced in PATKO BAT. i , Electron microscopy shows altered mitochondrial morphology in PATKO BAT. Representative images from 4 biologically independent samples. j , Oxygen consumption rate (OCR) of mitochondria isolated from BAT (n = 6). a - j , Biologically independent samples. Representative results from two ( a - c , j ) or three ( d , h ) independent experiments. Data presented as mean ± s.e.m. * p <0.05, ** p <0.01, *** p <0.001 vs. WT determined by two-tailed Student’s t-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Binding Assay, Gene Expression, Electron Microscopy, Isolation, Two Tailed Test

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: Brown adipose tissue from chow-fed WT and mutant mice housed at 30°C was analyzed. a , Levels of succinyl-CoA, glycine, and aminolevulinic acid (ALA) in BAT quantified using targeted metabolomics (n = 5 biologically independent samples per group). b , PATKO mice show reduced expression of Alas1 and Alas2 in BAT (n = 3 biologically independent samples per group). c , Nuclear labile heme is significantly lower in BAT of fat-specific PGRMC1/2 DKO mice housed at 30°C (n = 4 biologically independent samples per group). Similar to what is seen in PATKO mice, BAT of PGRMC1/2 DKO mice is discolored. Representative results from two independent experiments. d , Expression of REV-ERBα ( Bmal1 ) and BACH1 ( Fth1 ) targets in BAT of PATKO mice housed at 30°C (WT n = 5; PATKO n = 6). e , Circadian oscillation of clock components is not altered in PATKO BAT (n = 3 biologically independent samples per group per time point). f , GO category analysis (biological process) of significantly downregulated genes in RNAseq analysis of BAT from WT and PATKO mice housed at 30°C (n = 4 biologically independent samples per group). p values determined by standard accumulative hypergeometric statistical test. g , Circos plot of heme-related differentially-regulated genes (DEGs) showing that the majority (28/45) of them belong to the top 3 downregulated biological processes. Number in parenthesis below each biological process represents the total number of DEGs in PATKO BAT in that category. Blue lines refer to downregulated DEGs and red lines to upregulated DEGs. Data presented as mean ± s.e.m. * p <0.05, ** p <0.01, *** p <0.001 vs. WT determined by two-tailed Student’s t-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Mutagenesis, Expressing, Two Tailed Test

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: a - b , Inhibition for 48 hr of endogenous heme synthesis with 0.5 mM succinylacetone (FBS +SA), but not exogenous heme depletion (Heme-depleted FBS), in WT primary brown adipocytes phenocopies the mitochondrial defects of PATKO cells ( a , n = 8; b , n = 4). c - d , Treatment with SA (0.5 mM) dramatically reduces Ucp1 mRNA and protein levels (n = 3). e , Exogenous hemin (20 μM) does not correct mitochondrial dysfunction in PATKO cells (n = 3). f , PATKO brown adipocytes show higher levels of Rev-Erbα and BACH1 protein. g , Dual knockdown of Rev-Erbα and BACH1 in mature PATKO adipocytes restores mitochondrial respiration (n = 5). h , Pgrmc2 , Rev-Erbα , and Bach1 mRNA in control and knockdown cells. a-h , Biologically independent samples. Representative results from two independent experiments. Data presented as mean ± s.d. ** p <0.01, *** p <0.001 vs. WT; ### p <0.001 vs. Scramble determined by two-way ANOVA with multiple comparisons and a Bonferroni’s post-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Inhibition, Knockdown, Control

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: WT and PATKO mice were fed HFD for 20 weeks. a , H&E stain images of BAT from WT and PATKO mice fed HFD show similar morphology. Insets are magnified on the right. Scale bar is 100 μm. Representative images of 7 biologically independent samples. b , Gene expression analysis in BAT shows reduced levels of Fth1 and Bmal1 , targets of BACH1 and Rev-Erbα respectively, in PATKO BAT (WT n = 7; PATKO n = 8). c , H&E stain images of iWAT and eWAT from WT and PATKO mice fed HFD do not show clear differences. Scale bar is 100 μm. Representative images of 7 biologically independent samples. d , Size analysis of iWAT and eWAT adipocytes from HFD-fed WT and PATKO mice. X axis indicates μm 2 (n = 5 images of biologically independent samples). e , Gene expression analysis in iWAT reveals a modest increase in expression of genes involved in lipid handling. Similar to BAT, Bmal1 expression is significantly reduced in iWAT of PATKO mice (WT n = 7; PATKO n = 9). a-e , Biologically independent samples. Data presented as mean ± s.e.m. * p <0.05, ** p <0.01, *** p <0.001 vs. WT determined by two-way ANOVA with multiple comparisons and a Bonferroni’s post-test.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Staining, Gene Expression, Expressing

Journal: Nature

Article Title: PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function

doi: 10.1038/s41586-019-1774-2

Figure Lengend Snippet: Model of the proposed role for PGRMC2 in heme dynamics in brown adipocytes. PGRMC2 acquires heme from PGRMC1, which forms a complex with FECH, the last enzyme in heme synthesis. PGRMC2, located in the ER and the nuclear envelope, facilitates delivery of labile heme to the nucleus. Nuclear labile heme alters expression of genes regulated by heme-responsive transcriptional repressors, such as REV-ERBα and BACH1, that impact mitochondrial bioenergetics. Also shown are FVLCR1b, a mitochondrial heme exporter identified in erythrocytes, and HRG-1, a plasma-membrane heme importer characterized in macrophages. FVLCR1b and HRG-1 are both expressed in brown adipocytes, but their role in heme dynamics in this cell type remains to be defined.

Article Snippet: The antibodies and dilutions used in this work were: PGRMC2 (1:1,000, Bethyl Laboratories, A302-954A and A302-955A), PGRMC1 (1:1,000, Bethyl Laboratories, A304-561A), PPARγ, EV-ERBα (1:200, Santa Cruz Biotechnology, sc-7273 and sc-100910), BACH1 (1:500, R&D Systems, AF5777), UCP1 and OxPhoS (1:5,000 and 1:300, Thermo Fisher Scientific, PA124894 and 458099), GAPDH, TUBULIN, and HSP90 (1:5,000, GeneTex, GTX627408, GTX27291, and GTX101423), and CEBPδ (1:1,000, Abgent, AP20492c).

Techniques: Expressing, Clinical Proteomics, Membrane