Journal: Breast Cancer Research and Treatment

Article Title: Differential response of luminal and basal breast cancer cells to acute and chronic hypoxia

doi: 10.1007/s10549-023-06863-w

Figure Lengend Snippet: Chronic hypoxia affects metabolism in luminal breast cancer cells. A, B Heatmap of genes involved in glycolysis (A) and cytoskeleton (B) pathways in 24 h (acute) and 5 days (chronic) hypoxia in MCF-7 and HCC1143 cells. NA, not available; ****padj < 0.0001; ***padj < 0.001; **padj < 0.01; *padj < 0.05; ns, not significant. White color means that genes were filtered out in targeted RNA sequencing data after filtering by DESeq2 package in R software. C Lactate levels (mM) in 6 cell lines under normoxia normalized to the OD value determined in SRB assay. D, E Lactate levels measured in three luminal (MCF-7, T47D, BT474) (D) and 3 basal A (HCC1143, SUM149PT, HCC1806) (E) breast cancer cell lines under acute normoxia/ hypoxia and chronic normoxia/ hypoxia normalized to the OD value determined in SRB assay. F CA9 RNA expression level under hypoxia in luminal and basal A cell lines detected by qRT-PCR. Log 2 (2^(-ΔΔCT)) was calculated by normalizing to normoxia in each cell line. Error bars indicate SD for triplicate measurements. *** p < 0.001; ** p < 0.01; * p < 0.05. G GAPDH and PLOD2 protein expression detected by Western blot. B-actin serves as loading control. H Quantification of GADPH signal normalized to B-actin with Image J. Error bars indicate SD for triplicate measurements. ** p < 0.01; * p < 0.05; ns, not significant

Article Snippet: Membranes were blocked with 5% BSA and incubated with Carbonic Anhydrase IX (CA9) antibody (5649S; Cell Signaling Technology, Danvers, MA, USA), PLOD2 antibody (MAB4445; R&D Systems, Minneapolis, MN, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (sc-32233; Santa Cruz, Dallas, TX, USA), or β-actin antibody (sc-47778; Santa Cruz) overnight at 4 °C.

Techniques: RNA Sequencing, Software, Sulforhodamine B Assay, RNA Expression, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Breast Cancer Research and Treatment

Article Title: Differential response of luminal and basal breast cancer cells to acute and chronic hypoxia

doi: 10.1007/s10549-023-06863-w

Figure Lengend Snippet: Chronic hypoxia affects cell migration in basal breast cancer cells. A, B Cell tracking of MCF-7 and HCC1143 cell lines in acute (24 h) and chronic hypoxia (5 days) (A) , and calculated migration speed (B) . Error bars indicate SD for triplicate measurements. **** p < 0.0001; ** p < 0.01; ns, not significant. The pixels of acute hypoxia are 20 and of chronic hypoxia are 50 for cell tracking. C PLOD2 mRNA expression level detected by qRT-PCR in MCF-7 (two biological replicates) and HCC1143 cells (three biological replicates) comparing chronic hypoxia to normoxia. Error bars indicate SD for triplicate measurements. ** p < 0.01. D, E PLOD2 mRNA expression level in MCF-7 (D) and HCC1143 (E) cells under chronic hypoxia in presence of PLOD2 siRNA or control “kinasepool” siRNA (siKP). Average and SD from two biological replicates are shown. F Lactate levels measured for the indicated cell lines under chronic hypoxia normalized to the OD value determined in SRB assay. G, H Migration speed (G) and cell tracking (20 pixels) (H) analyzed for the indicated cell lines under chronic hypoxia. ns, not significant. Error bars indicate SD for triplicate measurements. ns, not significant

Article Snippet: Membranes were blocked with 5% BSA and incubated with Carbonic Anhydrase IX (CA9) antibody (5649S; Cell Signaling Technology, Danvers, MA, USA), PLOD2 antibody (MAB4445; R&D Systems, Minneapolis, MN, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (sc-32233; Santa Cruz, Dallas, TX, USA), or β-actin antibody (sc-47778; Santa Cruz) overnight at 4 °C.

Techniques: Migration, Cell Tracking Assay, Expressing, Quantitative RT-PCR, Control, Sulforhodamine B Assay