Journal: Iranian Journal of Biotechnology

Article Title: Downregulation of HMGB1 by miR-103a-3p Promotes Cell Proliferation, Alleviates Apoptosis and in Flammation in a Cell Model of Osteoarthritis

doi: 10.30498/IJB.2020.129470.2255

Figure Lengend Snippet: Effects of miR-103a-3p overexpression in LPS-induced chondrocytes. Cells were transfected with miR-103a-3p mimics or miR-NC for 48 h and then treated with LPS for 6 h. (A) MTT assay of LPS-induced chondrocytes. (B) Cell apoptosis was detected by Flow cytometry assay. (C) The relative protein levels of PCNA, caspase-3, and PARP were detected by western blot assay. (D) The production of IL-1β, IL-6, IL-10 and TNF-α in the cell culture supernatants was detected by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with control; # p < 0.05, ## p < 0.01, as compared with LPS+miR-NC

Article Snippet: After blocking with 2 % skim milk at 37 °C for 1 h, the membrane was incubated with primary antibodies against PCNA (1:1000, #4578, Cell signaling Technology, MA, USA), caspase-3 (1:1000, #9661, Cell signaling Technology, MA, USA), PARP (1:1000, #9542, Cell signaling Technology, MA, USA), HMGB1 (1:500, #9213, Cell signaling Technology, MA, USA) and GAPDH (1:500000, #10494-1-AP, Proteintech Group, Inc. IL, USA) at 4 °C overnight.

Techniques: Over Expression, Transfection, MTT Assay, Flow Cytometry, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer science

Article Title: Expression of heme oxygenase-1 in human leukemic cells and its regulation by transcriptional repressor Bach1.

doi: 10.1111/j.1349-7006.2010.01550.x

Figure Lengend Snippet: Fig. 1. Differential expression of heme oxygenase (HO)-1, nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and BTB and CNC homology 1 (Bach1) proteins in primary monocytes and leukemic cells. (A) Expression levels of HO-1, Nrf2, and Bach1 were determined by immunoblotting techniques in: whole-cell lysates of leukemic cell lines (U937, HL60, and K562); circulating leukemic cells from patients with AML (M1, n = 2; M2, n = 2; and M4, n = 3) or ALL (n = 4); and peripheral monocytes from healthy donors (n = 4). (B) Data are given as the mean ± SEM; the U937 protein level was assigned a value of 1. Figure key describes: AML, cells obtained from 12 AML patients (M1, n = 3; M2, n = 5; and M4, n = 4); Cell line, myelogenous leukemic cell lines U937, HL60, and K562; Monocyte, monocytes from four healthy donors. **P < 0.01 by paired t-test. (C) Fractionated cytoplasmic (Cyto) and nuclear (Nuc) extracts from AML cells (M1, patient #8) and CD14+ monocytes were analyzed separately. Actin and Lamin A ⁄ C served as endogenous loading controls for cytoplasmic and nuclear proteins, respectively.

Article Snippet: Human HO-1 siRNA and non-targeting negative control RNA were purchased from Ambion (Austin, TX, USA). (11) Human Bach1 siRNA and control RNA were obtained from Santa Cruz Biotechnology.

Techniques: Quantitative Proteomics, Derivative Assay, Expressing, Western Blot

Journal: Cancer science

Article Title: Expression of heme oxygenase-1 in human leukemic cells and its regulation by transcriptional repressor Bach1.

doi: 10.1111/j.1349-7006.2010.01550.x

Figure Lengend Snippet: Fig. 2. Regulation of heme oxygenase (HO)-1 expression by nuclear factor erythroid-derived 2 related factor 2 (Nrf2) and BTB and CNC homology 1 (Bach1) levels in the nuclei in response to LPS. (A) Expression levels of HO-1, Nrf2, and Bach1 in U937 cells (left) and CD14+

Article Snippet: Human HO-1 siRNA and non-targeting negative control RNA were purchased from Ambion (Austin, TX, USA). (11) Human Bach1 siRNA and control RNA were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Derivative Assay

Journal: Cancer science

Article Title: Expression of heme oxygenase-1 in human leukemic cells and its regulation by transcriptional repressor Bach1.

doi: 10.1111/j.1349-7006.2010.01550.x

Figure Lengend Snippet: Fig. 5. Regulation of heme oxygenase (HO)-1 expression by manipulation of the BTB and CNC homology 1 (Bach1) gene. (A) Expression levels of Bach1 and HO-1 were examined in U937 cells transfected with 2 lg Bach1-encoding pcDNA3.1+ plasmid (pBach1) or control plasmid (pCont) for 6 h. Densitometrical analysis of three independent experiments is shown in the right panel (mean ± SEM). (B) After U937 cells were incubated with 10 or 30 nM Bach1 siRNA or negative control RNA for 16 h, Bach1 and HO-1 levels were examined by immunoblotting. The blot was analyzed after longer exposure to detect small amounts of Bach1. (C) After U937 cells were treated with 30 nM Bach1 or control RNA for 16 h, then with PMA for a further 24 h, they were further treated with or without LPS (1 lg ⁄ mL) for 16 h, followed by analysis of the HO-1 and Bach1 levels. Densitometric analysis of the blots is shown in the right panel. Values are given as the mean ± SEM of HO-1 or Bach1 levels relative to actin levels from four independent experiments.

Article Snippet: Human HO-1 siRNA and non-targeting negative control RNA were purchased from Ambion (Austin, TX, USA). (11) Human Bach1 siRNA and control RNA were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Incubation, Negative Control, Western Blot

Journal: Cancer science

Article Title: Expression of heme oxygenase-1 in human leukemic cells and its regulation by transcriptional repressor Bach1.

doi: 10.1111/j.1349-7006.2010.01550.x

Figure Lengend Snippet: Fig. 4. BTB and CNC homology 1 (Bach1)-dependent transcriptional suppression of heme oxygenase (HO)-1 expression at Maf-recognition elements in PMA ⁄ LPS-treated U937 cells. (A) HO-1 mRNA levels were monitored by real-time PCR at the indicated time points after LPS (1 lg ⁄ mL) stimulation of PMA-treated (dotted line) or untreated (solid line) U937 cells. The expression level was standardized relative to the amounts of 18S rRNA. Values indicate relative quantity; the starting points of individual experimental series were assigned a value of 1. Data are given as the mean ± SEM for five independent experiments. **P < 0.01; *P < 0.05 by Kruskal–Wallis test. (B) HO-1 mRNA expression was determined in PMA-treated U937 cells before and after stimulation with LPS for 2.5 h in the presence or absence of actinomycin D (Act D; 5 lg ⁄ mL). Data are given as the mean ± SEM for three independent experiments; the basal expression level was assigned a value of 1. *P < 0.05 by the paired t-test. (C) PMA-treated U937 cells were transfected with 2 lg plasmid pGL3 HO-1(-4.5k), pGL3 HO-1(-4.0k), or pGL3 basic vector, mixed with 50 ng PRL-CMV vector. Luciferase activity was determined after stimulation with LPS for a further 12 h. Values are given as the mean ± SEM of relative luciferase activity for four independent experiments. The unstimulated pGL3 HO-1(-4.5k) samples were assigned a value of 1. (D) PMA-treated U937 cells were stimulated with LPS for the indicated periods of time (0–6 h). Bach1 levels were examined in cytoplasmic and nuclear fractions by immunoblotting. Actin and Lamin A ⁄ C served as endogenous loading controls for cytoplasmic (Cyto) and nuclear (Nuc) proteins, respectively.

Article Snippet: Human HO-1 siRNA and non-targeting negative control RNA were purchased from Ambion (Austin, TX, USA). (11) Human Bach1 siRNA and control RNA were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot

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: Antioxidants

Article Title: HO-1 Limits the Efficacy of Vemurafenib/PLX4032 in BRAF V600E Mutated Melanoma Cells Adapted to Physiological Normoxia or Hypoxia

doi: 10.3390/antiox11061171

Figure Lengend Snippet: Immunoblot analysis of pERK ( A ), total ERK ( B ), Bach1 ( C ) and HO-1 ( D ). MeOV-1 cells were adapted for 5 days to 18 kPa, 5 kPa or 1 kPa O 2 and then treated with 10 µM PLX4032 (or 0.05% DMSO) for 24 h. β-actin was used as loading control. Immunoblots are representative of 3–4 independent experiments. Data denote mean ± S.E.M., n = 3–4 independent cell cultures, § p < 0.001 vs. CTR; ★ p < 0.001 vs. DMSO; ★★ p < 0.01 vs. CTR; # p < 0.05 vs. DMSO; ## p < 0.01 vs. DMSO.

Article Snippet: After blotting, PVDF membranes (GE Healthcare, Amersham Place, UK) were blocked in non-fat dry milk (5% w / v in 1X tTBS) and then incubated with antibodies against ERK, p-ERK (1:1000, rabbit polyclonal antibody, Cell Signaling Technology, Leiden, The Netherlands), HO-1 (1:2000, rabbit polyclonal antibody, ORIGENE, Herford, Germany), Bach1 (1:4000, rabbit polyclonal antibody, Bethyl Lab, Montgomery, TX, USA), and HIF-1α (1:1000, rabbit polyclonal antibody, Abcam, Cambridge, UK).

Techniques: Western Blot

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: BACH1 was mainly located in the odontoblast layer of human dental pulp tissues. a Immunohistochemical staining was performed to show the BACH1 expression in healthy human dental pulp tissues; arrowheads indicate BACH1 expression. Scale bars = 50 µm. b BACH1 (red) and HO-1 (green) expression was detected by double immunofluorescence staining. Blue, DAPI-stained cells. c Positive cell rate analysis indicated that the expression of BACH1 and HO-1 increased in the odontoblast layer (red) compared with the cell rich zone (yellow). Scale bar = 20 µm. Data were presented as the mean ± standard error of the mean (n = 7). ** P < 0.01, *** P < 0.001

Article Snippet: BACH1 (14018-1-AP), HO-1 (66743-1-Ig), Lamin B1 (66095-1-Ig), α-Tubulin (66031-1-Ig), CoraLite488-Goat-anti-Rabbit IgG (H + L) and CoraLite594-goat-anti-Mouse IgG (H + L) were purchase from Proteintech (Chicago, IL, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Double Immunofluorescence Staining

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: The expression of BACH1 was changed during the odontoblastic differentiation of hDPSCs. a Changes in the mRNA level of BACH1 , DMP1 and DSPP on OM days 0,1,3,7 and 14. GAPDH was used as an internal control. b Changes in the total protein level of BACH1, DMP1 and DSPP on OM days 0, 1, 3, 7 and 14. Quantification of the relative protein expression via Image Pro Plus software, in which GAPDH was served as the internal control. c BACH1 protein expression of nuclear fraction on OM days 0, 1, 3, 7 and 14 had been tested by Western blot analysis. Lamin B1 was used as loading control. d Representative immunofluorescent labelling images are displayed. BACH1 (red), DAPI (blue) and Phalloidin (green) were imaged to show the subcellular distribution of BACH1 on OM days 0, 1 and 14. Scale bar = 10 µm. Data were presented as the mean ± standard error of the mean (n = 3). Statistically significant compared to the day 0 group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The gels in b, c were cropped, full-length gels are presented in Additional file : Original Band of Western blot Analysis Figs. S1 and S2. In Fig. 3b, the bands of BACH1 and GAPDH were from the same gel, while the bands of DSPP and DMP1 were from different gels. In Fig. 3c, the bands of BACH1 and Lamin B1 were from the same gel

Article Snippet: BACH1 (14018-1-AP), HO-1 (66743-1-Ig), Lamin B1 (66095-1-Ig), α-Tubulin (66031-1-Ig), CoraLite488-Goat-anti-Rabbit IgG (H + L) and CoraLite594-goat-anti-Mouse IgG (H + L) were purchase from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Control, Software, Western Blot

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: The downregulation of BACH1 impeded the proliferation and induced cell cycle arrest of hDPSCs in vitro. a The effect of BACH1 silencing on the cell proliferation of hDPSCs was determined by CCK-8 assay. BACH1 silencing group (LV-shBACH1) and control group (LC-NC). b EdU assay for cell proliferation. Red, EdU-positive cells; Blue, Hoescht-stained cells. The percentage of EdU-positive cells in the LV-shBACH1 and LC-NC was recorded. Scale bar = 50 µm. c Percentage of cell cycle distribution tested by flow cytometry. Statistically significant compared to the LV-NC group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: BACH1 (14018-1-AP), HO-1 (66743-1-Ig), Lamin B1 (66095-1-Ig), α-Tubulin (66031-1-Ig), CoraLite488-Goat-anti-Rabbit IgG (H + L) and CoraLite594-goat-anti-Mouse IgG (H + L) were purchase from Proteintech (Chicago, IL, USA).

Techniques: In Vitro, CCK-8 Assay, Control, EdU Assay, Staining, Flow Cytometry

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: BACH1 affected the odontoblastic differentiation of hDPSCs in vitro. a Transduced hDPSCs were cultured with OM for 7 days. ALP activity was evaluated by ALP staining and ALP activity assay in the LV-NC and LV-shBACH1 group. b hDPSCs were cultured with OM for 21 days. Calcium nodule deposition was evaluated by ARS staining assay. Scale bar = 200 µm. c mRNA level of BACH1 , HMOX-1 and odontogenic markers in LV-NC and LV-shBACH1 hDPSCs on OM 14 days. GAPDH was used as an internal control. d Total protein level of BACH1, HO-1 and odontogenic markers in LV-NC and LV-shBACH1 hDPSCs on OM 14 days. Quantification of the relative protein expression via Image Pro Plus software. GAPDH was used as an internal control. Data were presented as the mean ± standard error of the mean (n = 3). Statistically significant compared to the LV-NC group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The gels in d were cropped, full-length gels are presented in Additional file Original Band of Western blot Analysis Fig. S3. The bands of BACH1, DSPP, HO-1 and GAPDH were from the same gel, while the bands of RUNX2 and DMP1 were from different gels

Article Snippet: BACH1 (14018-1-AP), HO-1 (66743-1-Ig), Lamin B1 (66095-1-Ig), α-Tubulin (66031-1-Ig), CoraLite488-Goat-anti-Rabbit IgG (H + L) and CoraLite594-goat-anti-Mouse IgG (H + L) were purchase from Proteintech (Chicago, IL, USA).

Techniques: In Vitro, Cell Culture, Activity Assay, Staining, ALP Activity Assay, Control, Expressing, Software, Western Blot

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: The downregulation of BACH1 impaired the odontoblastic differentiation of hDPSCs was HO-1-independent. a Transduced hDPSCs were cultured for 7 days. ALP activity was evaluated by ALP staining and ALP activity assay in the LV-NC, LV-shBACH1 and LV-shBACH1 + SnPP group. b Total protein level of odontogenic markers in LV-NC, LV-shBACH1 and LV-shBACH1+ SnPP hDPSCs on 14 days. Quantification of the relative protein expression of odontogenic markers via Image Pro Plus software. GAPDH was used as an internal control. Statistically significant compared to the control group. c hDPSCs were cultured for 21 days. Calcium nodule deposition was evaluated by ARS staining assay. Scale bar = 1 mm. SnPP, Tin-protoporphyrin IX. hDPSCS were cultured in OM or OM + SnPP (10 µM). Data were presented as the mean ± standard error of the mean (n = 3). Statistically significant compared to every other group. * P < 0.05, ** P < 0.01, *** P < 0.001. The gels in b were cropped, full-length gels are presented in Additional file Original Band of Western blot Analysis Fig. S4. The bands of DSPP, RUNX2 and GAPDH were from the same gel, while the band of DMP1 was from other gel

Article Snippet: BACH1 (14018-1-AP), HO-1 (66743-1-Ig), Lamin B1 (66095-1-Ig), α-Tubulin (66031-1-Ig), CoraLite488-Goat-anti-Rabbit IgG (H + L) and CoraLite594-goat-anti-Mouse IgG (H + L) were purchase from Proteintech (Chicago, IL, USA).

Techniques: Cell Culture, Activity Assay, Staining, ALP Activity Assay, Expressing, Software, Control, Western Blot