Fig. 6: p38γ silencing decreases Rb phosphorylation, cyclin E1/A expression and disrupts mitochondrial functions in NPC cells.

Co-immunoprecipitation (Co-IP) assay tested the association between p38γ, CDK2 and Rb (A) in CNE-1 cells. Their expression was presented in “Input” (A). The stable CNE-1 cells expressing the p38γ-shRNA-s1 (“p38γ-shRNA”), the lenti-CRISPR/Cas9-p38γ-KO construct (“p38γ-KO”), or the scramble control shRNA plus the lenti-CRISPR/Cas9-control construct (“shC+Cas9-C”) were established, expression of listed proteins and mRNAs was shown (B–D and P); Cells were further cultured for applied time periods, mitochondrial depolarization (by measuring JC-1 green monomer intensity, M), ROS contents (by measuring CellROX intensity, N), ssDNA contents (O) and ATP contents (Q) were tested. CNE-1 cells expressing the lentiviral p38γ-expressing construct (“OE-p38γ-L1/L2”) or the empty vector (“Vec”) were established, expression of listed proteins was shown (E). CNE-1 cells were treated with the p38γ specific pharmacological inhibitor pirfenidone (PFD, 0.5 mg/mL) or the vehicle control (0.1% DMSO, “Veh”) for indicated time periods, expression of listed proteins in total cell lysates (F) and mitochondrial fraction lysates (L) were shown; Cell proliferation was tested by EdU staining assays (G). OE-p38γ-L1 CNE-1 cells were further transduced with A non-phosphorylated Rb mutant construct (“Rb-mut”) or the empty vector (“Vec”), expression of listed proteins was shown (H); Cells were further cultured for 72 h and cell proliferation was tested by measuring EdU-positive nuclei ratio (I). The Compartments Database shows the subcellular localization of p38γ protein (J). The confocal fluorescence images showed p38γ protein (in green fluorescence) and MitoTracker Orange (in orange fluorescence) co-localization in CNE-1 cells (K). “pare” stands for the parental control cells. *P < 0.05 vs. “pare”/“Vec” cells. Scale Bar = 100 μm (G, K, M and O).