Fig 1: Transcriptome analysis (and selected transcript and expression validation) of noninvaded versus invaded breast cancer cells in CIMMS.(A) Read counts were collected and averaged from invaded and noninvaded subpopulations of MDA-MB-231 cells harvested from five simple collagen I microgels (2.4 mg/ml) on day 4 after seeding at 400,000 cells/ml. The data are represented as a heat map of expression level (shown as variance-stabilized read counts per million, where yellow = high and blue = low) of the 244 genes (rows) determined to be significantly differentially expressed [with FDR < 0.05 based on the quasi-likelihood negative binomial generalized log-linear model (glmQLFit) from the edgeR package (51)] for noninvaded (“N avg.,” left column) and invaded cells (“I avg.,” right column). The white trace labeled “density” in the legend represents the distribution of the different expression levels in the dataset. Four genes (FPR1, BCL3, CX3CL1, and CDH1) are highlighted; the full list is found in table S1 (list 1). (B) ddPCR results for transcripts of FPR1 (top), BCL3 (mid-top), CX3CL1 (mid-bottom), and CDH1 (bottom) in noninvading (white bars) and invading (black bars) cells harvested from CIMMS experiments treated identically to (A), with error bars = 1 SD. Expression values for the four genes were normalized to the expression level of housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). (C) Top-view fluorescent confocal microscope images of microgels from CIMMS experiments treated identically to (A), with images in the left column stained blue for nuclei, images in the middle column immunostained green for FPR1 (top), BCL3 (mid-top), CX3CL1 (mid-bottom), and E-cadherin (bottom), and images in the right column overlaid. The white dashed lines indicate the edge of the gels. Scale bars, 50 µm.
Fig 2: MiR-627-5p is inhibited by hypoxia in HCC cells. (A) Heat map for hypoxia-induced genes Hep3B cells. Chip Analysis on Gene Expression was conducted in Hep3B cells exposed to normoxia (20% O2) or hypoxia (1% O2) for 24h. BCL3 and CCND1 mRNA expression were induced by hypoxia. The samples were triplicated. (B) Hep3B and SMMC7721 cells were exposed to normoxia (20% O2) and hypoxia (1% O2) for 12h, 24h and 48h. And miR-627-5p expression was determined by RT-qPCR analysis (mean ± SD; n = 3). *P < 0.05 vs. Normoxia (two-way ANOVA) (C) The HIF-1a or HIF-2a knockdown subclones of Hep3B and SMMC-7721 were exposed to 20% or 1% O2 for 24 hours, followed by analysis of miR-627-5p levels by RT-qPCR (mean ± SD; n = 3). ***P < 0.001 vs. NTC at 20% O2; ###P < 0.001 vs. NTC at 1% O2 (two-way ANOVA). (D) Hep3B and SMMC-7721 cells were exposed to 20% or 1% O2 for 16h, and ChIP assays were performed using the indicated antibodies. Primers encompassing candidate HIF binding sites located 7.0 kb 5'and 4.7 kb 5' to the MIR627 gene transcription start site were used for qPCR and results were normalized to the first lane (mean ±SD; n = 3). There was no significant change (Student's t test). (E) The oligonucleotide spanning HRE-1 site or HRE-2 site was inserted into the reporter plasmid pGL2-promoter respectively, in which a basal SV40 promoter drives firefly luciferase expression. Hep3B and SMMC-7721 cells were co-transfected with pGL2/MIR627-HRE and exposed to 20% or 1% O2 for 24h. The ratio of firefly/Renilla luciferase activity was analyzed (mean ± SD; n = 3). There was no significant change (Student's t test).
Fig 3: Hypoxia inhibits miR-627-5p expression by HIF-1a/HDAC3/miR-627-5p pathway in HCC. (A) HDAC3 knockdown or NTC subclones of Hep3B and SMMC-7721 were exposed to normoxia or hypoxia condition. And Western blot was applied to analyze the protein expression change of HDAC3. (B) RT-qPCR analysis was applied to test HDAC3 mRNA expression change in HDAC3 knockdown or NTC subclones of Hep3B and SMMC-7721 cells, which were exposed to normoxia or hypoxia (mean ±SD; n = 3). *P < 0.05, two-way ANOVA. Co-transfection of HIF-1a and pcDNA/HDAC3 plasmid or the corresponding controls was conducted in Hep3B (C) and SMMC-7721 (D) cells, and the subclones were exposed to normoxia or hypoxia. RT-qPCR analysis was applied to test miR-627-5p expression change (mean ±SD; n = 3). ***P < 0.001, two-way ANOVA. Hep3B (E) and SMMC-7721 (F) with miR-627-5p mimics or control were exposed to normoxia or hypoxia condition. And Western blot was applied to analyze the protein expression change of HIF-1a, BCL3 and CCND1.
Supplier Page from Abcam for Anti-Bcl3 antibody