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. 2021 Jul 7;16(7):e0241092. doi: 10.1371/journal.pone.0241092

Fig 4. PFKFB3-4 overexpression facilitates cell viability and proliferation.

Fig 4

(A) Quantification of PFKFB3-4+5 mRNA levels from HEK-293 (yellow) and U87 cells (grey) stably overexpressing PFKFB3-4 (OE -4), respectively. mRNA-measurement was done with 4 independent experiments (N = 4, n = 1–2). PFKFB3-4 mRNA quantity measured by quantitative PCR was normalized to the amount of TBP mRNA and compared to mock samples. (B) Western blot analysis to confirm the overexpression of PFKFB3-4 with polyclonal PFKFB3 antibody. β-Tubulin served as loading control, 10 μg protein was loaded per lane. 1 refers to PFKFB3-1 and 4 refers to PFKFB3-4. The ratio of protein bands (PFKFB3-4 to β-Tubulin) was calculated from the intensity values of protein bands (n = 3). (C) Effect of stable PFKFB3-4 overexpression on cell viability, measured by WST-1 assay. After a 4 h incubation period with WST-1 reagent the absorbance was measured at 450 nm/600 nm. WST-test was done in 4–5 independent experiments in 7–8 times analyses (N = 4–5, n = 7–8). (D) Effect of stable PFKFB3-4 overexpression on proliferation measured by BrdU-assay. After 20 h incubation period with BrdU, the absorbance was measured at 450 nm/ 600 nm. BrdU-test were done three times in 6–7 analysis (N = 3, n = 6–7). (E) Influence of stable overexpression of PFKFB3-4 on the mRNA levels of PFKFB3-1 and PFKFB3-11 compared to mock samples. PFKFB3 mRNA quantity measured as duplicate in three independent experiments (N = 3, n = 2) by quantitative PCR and was normalized to the amount of TBP mRNA and compared to mock samples. All values represent the mean ± SEM. All groups were compared using the paired t-test or Mann Whitney test.