The impact of LDH-A inhibition on HIF-1 activity.

The assessment of HIF-1 activity after 6 hours of different treatments (A). VEGF-A secretion (a downstream target of HIF-1) by 4T1-HRE A5NC and A2-10KD cells under different conditions over 6 hours (B). The BLI-based HIF-1 and ELISA-based VEGF-A measurements are normalized to cell number. Statistics: **** pHIF-1 activity in tumors (C-E). Bioluminescence imaging of HIF-1 reporter activity in 4T1-HREexGLuc-IRES2-GFP tumors was performed at week 1–2. Data are expressed as tumor-to-background ratios normalized to initial values of week 1; ±SEM (C). Analyses of percent HIF-1α positive cells and area of Pimonidazole staining were performed and quantified using MetaMorph software by thresholding images. A greater percent of HIF-1α positive cells (D) and area of pimonidazole staining are seen in 4T1-HRE A5NC tumors compared to the LDH-A knock-down 4T1-HRE A2-10KD and 4T1-HRE A3-8KD tumors (E). Two independent experiments were performed, and immunofluorescence staining was performed on different days and on different samples.