MCU controls melanoma progression through a redox-controlled phenotype switch (2022)
Figure 3

Figure 3. MCUA knockdown suppresses tumor growth and promotes invasion in vitro and in vivo.

(A-B) Proliferation of 1205Lu (A) and WM3734 (B) stable MCUA_KD lines over 72 h, shown as percent of the respective control (shCTRL at 24 h) (n≥7 biological replicates/condition/day).

(C) Representative images of the migrated stained 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) cells on the lower side of the insert.

(D) Quantification of the transwell migration in 1205Lu stable MCUA_KD lines, based on the number of stained cells (n=4 biological replicates/condition, shown also by individual data points).

(E) Representative images of 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm.

(F) Quantification of 1205Lu stable MCUA_KD spheroid core size (n=6 biological replicates/condition, shown also by individual data points).

(G) Representative images of WM3734 shCTRL (black frame), shMCU_1 (darker orange frame) and shMCU_2 (lighter orange frame) melanoma spheroids. Live cells are shown in green. Scale bar: 100 µm.

(H) Quantification of WM3734 stable MCUA_KD spheroid core size (n=5 biological replicates/condition, shown also by individual data points).

(I) Invasion potential of WM3734 shCTRL (black), shMCU_1 (darker orange) and shMCU_2 (lighter orange) over a period of 10 days (n≤4 biological replicates/condition/day, shown also by individual data points).

(J-K) Migration potential of 451Lu and 451Lu BRAF inhibitor-resistant (451Lu-BR3) (J) and WM983B and WM983B BRAF inhibitor-resistant (WM983B-BR) cells (K) over 24 h (n=3 biological replicates/condition, shown also by individual data points).

(L) Representative images of 451Lu wild-type (grey frame), 451Lu-BR3 (lighter green frame), WM983B wild-type (grey frame) and WM983B-BR (darker green frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm.

(M-N) Quantification of 451Lu versus 451Lu-BR3 (M) and WM983B versus WM983B-BR (N) spheroid core size (n≥9 biological replicates/condition, shown also by individual data points).

(O) Representative images of mice in the shCTRL group and the shMCU_1 group after 36 days of tumor growth (n=10 mice/group).

(P) Tumor volume growth over 36 days post-inoculation.

(Q) Average weight of the tumors at the end point (36 days) upon removal (individual values shown as points).

(R) Exemplary image of 1205Lu shCTRL (black frame) and shMCU (blue frame) metastases in mouse lung tissue sections; paraffin sections were made from the removed lungs; H&E staining was carried out and the sections were scanned using an Axio Scan.Z1 microscope. Scale bar: 100 µm.

(S) Quantitative evaluation of the incidence (shown in percent) of lung metastases, total amount of positive animals identified with metastases, total area of all metastases in a group and the average area per metastasis in shCTRL group (black) and shMCU group (blue).

Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).