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. 2022 Jun 3;25(7):104527. doi: 10.1016/j.isci.2022.104527

Figure 3.

Figure 3

Progesterone-induced cell death is at least partly mediated by the activation of p38

(A) LLC1 tumor cells were incubated with Prog (50 μM) or the vehicle (Veh) for the indicated timepoints. Lysates were then assessed for evidence of MAPK activation by Western blot. The vertical gaps between the data for Veh and Prog for each antibody indicate separate blots; blots were exposed to the same X-ray film to ensure equal exposure to chemiluminescent signals (Data S2). Reactivity of antibodies recognizing phosphorylated (P) and total (T) MAPKs are shown. Gapdh controls are also shown. Data for P-JNK, JNK, Gapdh (Veh and Prog), P-p38 (Prog), and p38 (Prog) are mirror images of the original (Data S2). Densitometry was performed on raw data.

(B) Densitometry analysis of data in Figure 3A. Each phospho (P) band at 20 min was normalized to its corresponding band indicating total (T) reactivity. Fold change in phosphorylation (Prog vs Veh) at 20 min is represented. Values represent arithmetic mean fold change ± SD (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001 by the unpaired t-test.

(C) LLC1 tumor cells were incubated with inhibitors (i) for ERK (PD98059), JNK (JNK II inhibitor), or p38 (SB203580) (20 μM) for 1 h followed by treatment with Prog (50 μM) for 20 min. Veh for i + Prog: DMSO + ethanol. Lysates were then assessed for evidence of MAPK activation by Western blot. Reactivity of antibodies recognizing phosphorylated (P) and total (T) MAPKs are shown. Gapdh was employed as control; one representative experiment is shown. The Veh (Prog) and Prog lanes for Gapdh have been used across this representation, as they comprise samples from the same experiment.

(D) Densitometry analysis of data in Figure 3C. Each phospho (P) band was normalized to its corresponding band indicating total (T) reactivity. Fold change in phosphorylation (Prog vs Veh) at 20 min is represented. Values represent arithmetic mean fold change ± SD (n = 3). ∗p < 0.05, ∗∗∗p < 0.001 by the unpaired t-test.

(E) Densitometry analysis of data in Figure 3C. Each phospho (P) band was normalized to its corresponding band indicating total (T) reactivity. Fold change in phosphorylation (i + Prog vs Prog) at 20 min is represented. Values represent arithmetic mean fold change ± SD (n = 3). ∗p < 0.05, ∗∗∗p < 0.001 by the unpaired t-test.

(F) LLC1 tumor cells were pre-incubated with indicated inhibitors (i, 20 μM) for 1 h followed by treatment with Prog (15 μM) for 24 h. Intracellular ATP was measured to assess cell viability. Data are plotted as percentage viability over untreated cells. Values represent arithmetic mean ± SD (n = 3). ∗∗∗p < 0.001 by the unpaired t-test.