Fig 1: A combination of Z734 and lapatinib inhibits the growth of breast cancer. (A) MCF–7 cells were incubated with Z734 (10 μM) and/or lapatinib (0.5 μM), and p–ERK2, ERK2, p–p53, p53 and HERC3 protein expressions were detected by WB. (B) Representative images of ERK2 and p53 immunofluorescence staining in MCF–7 treated with Z734 (10 μM) or lapatinib (0.5 μM). DAPI was used to stain the nuclei. Quantification of fluorescence was performed on the Average Optical Density using the ImageJ software. Scale bars = 10 μm. (C) ELISA was used to detect the expression levels of Ki67 in MCF–7 to reflect cell proliferation after Z734 and/or lapatinib treatment. (D) Caspase3 activities in overexpressing of ERK2 in MCF–7 treated with Z734 (10 μM) and/or lapatinib (0.5 μM) were detected. (E) MCF–7 were treated with Z734 (10 μM) and/or lapatinib (0.5 μM) for 12 h, followed by flow cytometry to detect cell apoptosis. (F) Migration of MCF–7 cells were evaluated using the transwell assay after being treated with Z734 (10 μM) and/or lapatinib (0.5 μM). Scale bars = 20 μm. (G) Body weights of female Balb/c mice with different treatment groups (n = 5 for Control group; n = 5 for Z734 group; n = 5 Lapatinib group; n = 5 for Z734 + Lapatinib group). (H) Picture (n = 5 for Control group; n = 5 for Z734 group; n = 5 Lapatinib group; n = 5 for Z734 + Lapatinib group, combined results of 2 independent experiments) of MCF–7 breast tumor across four groups. (I,J) The volumes (Figure 6I) and weights (Figure 6J) were measured and quantified (n = 5 for Control group; n = 5 for Z734 group; n = 5 Lapatinib group; n = 5 for Z734 + Lapatinib group). Data shown in (B–G,I,J) were used one–way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 2: Z734 causes apoptosis and inhibits MCF–7 cells proliferation and migration. (A) The chemical structure of Z734. (B) Apoptosis levels were detected via flow cytometry. WT MCF–7 cells were treated with Z734 (0, 5, 10, and 15 μM) for 12 h. Flow cytometric analysis of apoptotic cells was conducted using AnnexinV–FITC. (C) Measurement of Caspase3 activity after Z734 intervention in MCF–7 cells. (D) Apoptosis–related protein levels after treatment with Z734 were determined via Western blot analysis. (E) MCF–7 cells were treated with different doses of Z734 and incubated for 12 h. Cell proliferation was assessed via CCK–8 assays. (F) Ki67 distribution after treatment with Z734 was determined via immunofluorescence. Quantification of fluorescence was performed on the average optical density using the ImageJ software. Scale bars = 10 μm. (G) The percentage of colonies of MCF–7 cell lines was determined after treatment with Z734. (H) MCF–7 cells were treated with Z734 for 12 h and then analyzed for migration by transwell analysis. Scale bars = 20 μm. Data shown in (B,C,E,G,H) were used one–way ANOVA with Tukey’s post hoc test. Data shown in (F) was used two–tailed Student’s t–test. All assays were performed in triplicate, and the data are expressed as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., no significant difference.
Fig 3: Role of HMGB1 in human UC-associated carcinogenesis. Investigation of the role of HMGB1 in mucosal immunity and epithelial differentiation in biopsy specimens from five cases of UC-associated CRC. (A) Histological appearance of inactive mucosa (N), active mucosa (A), dysplasia (D), and cancer (C). Scale bar = 100 μm. (B–E) Protein levels assessed by ELISA. (B) HMGB1. (C) Ki67 expression (proliferation). (D) ALP and MUC2 levels (epithelial differentiation). (E) DEF and BA (intestinal flora maintenance). (F–H) Flow cytometry for activated CD8 (CD8+IFNγ+) (F), exhausted CD8 (CD8+PDL1+) (G) and naïve CD8 (CD8+CD62L+) (H). Error bars = standard deviation of five cases and three independent trials. Statistical differences were calculated using analysis of variance with Bonferroni correction. UC, ulcerative colitis; HMGB1, high-mobility group box-1; N, non-active mucosa; A, active UC mucosa; D, UC-related dysplasia; C, UC-related carcinoma; MUC2, mucin 2; ALP, alkaline phosphatase; αDEF, α-defensin; BA, butyric acid; IFNγ, interferon γ; PDL1, programmed cell death ligand-1.
Fig 4: Effects of HMGB1 on intestinal epithelial cells. Mouse IEC6 intestinal epithelial cells were treated with HMGB1 (20 μg/mL, 48 h) to assess the effects of HMGB1. (A) RAGE and TLR4 gene expression levels. (B) Cell growth analysis. (C) Mitochondrial stress assays. (D) OXPHOS parameters. (E) Glycolytic stress assay. (F) Ki67 (proliferation). (G) MUC2 and ALP expression (intestinal epithelial differentiation). (H) DEF (intestinal flora maintenance). Error bars: standard deviation from three independent trials. Statistical differences were calculated using analysis of variance with the Bonferroni correction. HMGB1, high-mobility group box-1; RAGE, receptor for advanced glycation end products; TLR4, Toll-like receptor 4; ACTB, β-actin; OXPHOS; oxidative phosphorylation; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; MUC2, mucin 2; ALP, alkaline phosphatase; αDEF, α-defensin.
Fig 5: Effects of HMGB1 on hyperplastic mucosa surrounding CRC. Investigation of the role of HMGB1 in mucosal immunity and epithelial differentiation using biopsy specimens from 10 cases of CRC with hyperplastic mucosa surrounding the cancer lesions. (A) Histological appearance of the mucosa distant from CRC focus (C) and the hyperplastic mucosa surrounding CRC focus (H). Scale bar = 100 μm. (B–E) Protein levels of HMGB1 (B), Ki67 (proliferation) (C), ALP and MUC2 levels (epithelial differentiation) (D), and DEF and BA (intestinal flora maintenance) (E). (F–H) Flow cytometry for activated CD8 (CD8+IFNγ+) (F), exhausted CD8 (CD8+PDL1+) (G) and naïve CD8 (CD8+CD62L+) (H). (I–K) Relationships between HMGB1 and activated CD8 cells (CD8+IFNγ+) (I), or exhausted CD8 (CD8+PDL1+) (J), or naïve CD8 (CD8+CD62L+) (K). (I–K) Dots represent each case of cancer (C) and hyperplastic mucosa (H). Error bars: standard deviation of 10 cases and three independent trials. Statistical differences were calculated using analysis of variance with Bonferroni correction. Regression analysis was performed using Spearman’s correlation test. HMGB1, high-mobility group box-1; C, control non-hyperplastic mucosa distant from cancer lesion (more than 5 cm); H, hyperplastic mucosa surrounding cancer lesion (within 1 cm); MUC2, mucin 2; ALP, alkaline phosphatase; αDEF, α-defensin; BA, butyric acid; IFNγ, interferon γ; PDL1, programmed cell death ligand-1.
Supplier Page from Abcam for Human Ki67 ELISA Kit