Fig. 3: TM4SF5-mediated ROS modulation resulted in increased survival in epithelial cells. | Cell Death & Disease

Fig. 3: TM4SF5-mediated ROS modulation resulted in increased survival in epithelial cells.

From: TM4SF5-mediated CD44v8-10 splicing variant promotes survival of type II alveolar epithelial cells during idiopathic pulmonary fibrosis

Fig. 3

a, c Cells stably transduced with control (−) or HA-TM4SF5 plasmid-containing retrovirus were transfected with NOX2 and NOX4 plasmids (a) and subsequently cultured under serum starvation (b) or TNF-α treatment (c) in parallel to normal control condition (Cont) followed by DCF-DA fluorescence staining for intracellular ROS measurement. P values were calculated by two-tailed, unpaired Student’s t-test. d Cells stably transduced with control (−) or HA-TM4SF5 retrovirus were treated with control vehicle (Control) or TNF-α, followed by fluorescence and phase-contrast imaging. e Mouse embryonic fibroblasts (MEFs) from wild-type (+ / +), heterozygous knockout (−/ +), or homozygous knockout (−/−) animals were treated with TNF-α before DCF-DA staining for ROS measurement. f Cells were stably transduced without (−) or with HA-TM4SF5 retrovirus with vehicle DMSO (−) or TSAHC treatment, and were concomitantly treated with TNF-α before ROS measurement. g Cells stably transduced without (−) or with HA-TM4SF5 retrovirus were transfected with control (−) or NOX2/NOX4 plasmids along with control (−) or siRNA against CD44v8-10 (#1 or #2 sequences, Table 1) before TNF-α treatment and ROS measurement. P values were calculated by two-tailed, unpaired Student’s t-test. h Cells were stably transduced with control (−) or shTM4SF5-encoding (#2 or #4 sequence of TM4SF5, Table 1) lentivirus and then transfected with control (−) or FLAG-CD44v8-10 plasmid before relative cell survival measurement via MTT assay. i NCI-H727 cells were treated with shRNAs for a control sequence or to suppress endogenous TM4SF5 (#2 or #4), NCI-H358 cells were transfected with control (−) or HA-TM4SF5 ( + ) plasmids for 48 h, and they were then incubated without (Control) or with TNF-α (2 ng/ml) for 6 h before determination of MitoSOX fluorescence. The p values were analyzed by ANOVA with Tukey’s range-test. P values <0.05 were considered statistically significant. Data represent three independent experiments. See also Figure S3

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