MCU controls melanoma progression through a redox-controlled phenotype switch (2022)
Figure 5

Figure 5. MCUA controls redox signaling in melanoma cells.

(A-C) Mitochondrial hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using mito-HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (A). Scale bar: 10 µm. Quantification of mito-HyPer ratio in 1205Lu (B) and WM3734 (C) under resting state (1205Lu ─ shCTRL: n=217 cells from 6 biological replicates; shMCU_1: n=197 cells from 7 biological replicates; shMCU_2: n=212 cells from 7 biological replicates; WM3734 ─ shCTRL: n=104 cells from 6 biological replicates; shMCU_1: n=107 cells from 7 biological replicates; shMCU_2: n=114 cells from 7 biological replicates).

(D-F) Cytosolic hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (D). Scale bar: 10 µm. Quantification of HyPer ratio in 1205Lu (E) and WM3734 (F) under resting state (1205Lu ─ shCTRL: n=232 cells from 6 biological replicates; shMCU_1: n=290 cells from 6 biological replicates; shMCU_2: n=290 cells from 6 biological replicates; WM3734 ─ shCTRL: n=443 cells from 9 biological replicates; shMCU_1: n=297 cells from 9 biological replicates; shMCU_2: n=233 cells from 9 biological replicates).

(G-H) Quantification of resting cytosolic H2O2 levels (HyPer) in WM3734 upon siRNA-mediated MCUA_KD (siCTRL: n=58 cells from 3 biological replicates; siMCU: n=68 cells from 3 biological replicates) (G) and MCUA overexpression (O/E) (CTRL: n=52 cells from 3 biological replicates; MCU O/E: n=66 cells from 3 biological replicates) (H).

(I-K) Mitochondrial glutathione redox potential, measured with mito-Grx1-roGFP2 in 1205Lu and WM3734 with and without stable MCUA_KD. Representative ratiometric images (F385 nm/F475 nm) are shown for all conditions (I). Scale bar: 10 µm. Quantification in 1205Lu (J) and WM3734 (K) under resting state (1205Lu ─ shCTRL: n=231 cells from 10 biological replicates; shMCU_1: n=176 cells from 7 biological replicates; shMCU_2: n=164 cells from 7 biological replicates; WM3734 ─ shCTRL: n=127 cells from 7 biological replicates; shMCU_1: n=142 cells from 7 biological replicates; shMCU_2: n=116 cells from 7 biological replicates).

(L-M) Resting mitochondrial hydrogen peroxide (H2O2; mito-HyPer) in 451Lu and BRAF inhibitor-resistant 451Lu-BR3 (L) and WM983B and BRAF inhibitor-resistant WM983B-BR (M) (451Lu: n=227 cells from 8 biological replicates; 451Lu-BR3: n=166 cells from 6 biological replicates; WM983B: n=193 cells from 8 biological replicates; WM983B-BR: n=208 cells from 9 biological replicates).

(N) Migration (4h) of 1205Lu wild-type cells upon 4 h pre-treatment with 1 mM BSO, 200 µM NAC and 1 mM BSO + 200 µM NAC (n=6 biological replicates/condition, shown also by individual data points).

(O) Migration (4h) of WM3734 wild-type cells upon 4 h pre-treatment with 100 µM H2O2, 200 µM NAC and 100 µM H2O2 + 200 µM NAC (n=4 biological replicates/condition, shown also by individual data points).

(P) Migration of 1205Lu shCTRL and shMCU untreated and shMCU pre-treated for 4 h with 100 µM H2O2 (n=6 biological replicates/condition, shown also by individual data points).

Data information: All data are presented as mean ± SEM. (A-F) and (I-M) were measured in Ringer's buffer containing 0.5 mM Ca2+ and (G-H) in Ringer's buffer containing 1 mM Ca2+. For (A-M) statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. For (N-O) statistical significance was determined using paired, one‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01. For (P) statistical significance was assessed using paired, two‐tailed Student's t‐test, (**) p < 0.01. In all statistical analyses, KD, O/E or treated cells were compared to their respective control.