Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells
Fig 5
Effects of HV1 KO on wound healing, H2O2 production, metabolism, and bioactive molecules.
(A) The quantitative analysis of the wound healing assay illustrated in B. The chart shows the percentage coverage of each wound by cells after 24 hr. Analysis was performed using ImageJ and the chart shows data from 3 experiments performed in triplicate. Data are not significantly different. (B) Images of wells 24 hours apart after wounding with a 200 μl pipette. The black marks are scratch marks to denote position. MDA-MB-231 cells treated with virus containing the sequence for Cas9, 4a, or 5f2 for HV1 knockout. Cells were kept in 1% FBS but with high glucose. (C) The release of H2O2 from MDA-MB-231 cells. The data is from 4 experiments performed in triplicate. 4a is significantly different from Cas9 as determined by a one-way ANOVA with Bonferroni's post-test p<0.05. (D) A representative experiment illustrating the release of H2O2 over one hour. (E) The extracellular acidification rate (ECAR) of HV1 KO in 2 knockout clones compared to WT MDA-MB-231, Cas9, SRC, 2.1 and 3.1 knockdowns. Extracellular acidification was increased by adding 25 mM glucose after an hour of starvation. 10 μM oligomycin was used to inhibit mitochondria and enhance glycolysis. 2-deoxyglucose (5 mM) inhibits glycolysis to reveal the contribution of glycolysis to overall extracellular acidification. Data are the mean ± SEM from 5 experiments performed in at least sextuplet. (F) Western blot total protein extracts from MDA-MB-231 cells showing the different levels of protein expression or protein phosphorylation between WT and Cas9 expressing MDA-MB-231 cells and 3 different HV1 KO clones (1fb, 4a and 5f2).