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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Cancer Res. 2021 Mar 8;81(11):2903–2917. doi: 10.1158/0008-5472.CAN-20-3371

Figure 5. Specific inhibition of PIKfyve and p38MAPK activities are responsible for the effects on cellular viability and autophagy.

Figure 5.

(A) SW480 cells cultured for 3 days with 0.125 μM WX8 and/or 5 μM SB202190 with or without changing the medium every day to resupply the inhibitors. The amount of LC3-II form was determined by immunoblot, quantified, and presented as a percentage of the amount in vehicle-treated cells after normalization by histone content (B). The number of cells at day 3 was reported as a percentage of the cells cultured with vehicle (C). Values are mean ±SEM (n = 3).

(D) SW480 cells cultured for 12 hours with 0.125 μM WX8 and/or 5 μM SB202190, 30 μM Chloroquine was added as indicated and the cells collected 8 hours later. The amount of LC3-II form was determined by immunoblot, quantified and presented as a percentage of the amount in vehicle-treated cells after normalization by histone content (E).

SW480 and Hs27 cells were cultured for 3 days with the indicated inhibitors and the number of cells cultured with inhibitor reported as a percentage of the cells cultured with vehicle. (F) PIKfyve inhibitors (0.125 μM WX8, 0.3 μM NDF, 0.25 μM Vacuolin 1, 30 nM Apilimod, 0.6 μM YM201636) were applied either alone (individual inhibitors) or together with 5 μM SB202190. (G) p38MAPK inhibitors (5 μM SB202190, 5 μM LY2228820, 5 μM Skepinone-L, 3.5 μM TAK-715, 7.5 μM BIRB-796) were applied either alone or together with 0.125 μM WX8. “Synergy above additive” was calculated as in Fig. 3. Values are mean ±SEM (n = 3).

(H,I) p38MAPK in A549 cells was depleted with the indicated concentration of p38MAPK siRNA or control siRNA and incubated with 0.375 μM WX8. Cells were collected after 3 days, and the counts plotted as a percentage of the control siRNA-treated cells. Values are mean ±SEM (n = 3).

(J) The amount of p62 protein was determined by immunoblot of cells cultured for 1 or 3 days with the indicated inhibitors.

A375 cells, stably expressing wild-type or N1939K mutant PIKfyve protein, or vector transduced, were cultured for 3 days in the presence of the indicated concentration of WX8 (K), a set of autophagy inhibitors (3.75 μM WX8, 6 μM NDF, 5 μM Vacuolin-1, 0.3 μM Apilimod, 12.5 μM YM201636, 30 μM chloroquine) (L) or WX8 and SB202190 (M). The cells were collected and the counts presented as a percentage of the vehicle-treated cells. The vehicle was set to 0.01 μM instead of 0 μM. Values are mean ±SEM (n = 3).