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. 2022 May 25;14(2):375–403. doi: 10.1016/j.jcmgh.2022.05.006

Figure 6.

Figure 6

SMYD5 negatively regulates PGC-1α at the post-transcriptional level. (A) Representative images (left) and quantitative analysis (right) of IF staining of SMYD5 and PGC-1α expression in HCT116 cells with either SMYD5 OE or KO. Isotope controls with no primary antibodies also are shown (left, bottom panel). Scale bars: 25 μm. ∗P < .05, n = 3. (B) Representative Western blots (top) and quantitative analysis (bottom) of SMYD5 and PGC-1α expression (normalized to GAPDH) in SMYD5 OE or KO HCT116 cells. The expression levels of SMYD5 and PGC-1α in parental HCT116 cells were set as 1. ∗∗∗P < .001, comparison of PGC-1α levels among groups; n = 3. (C) Representative IF staining (left) and quantitative analysis (right) of SMYD5 (red staining indicated by white arrowheads) in intestinal mucosa epithelia of Smyd5fl/fl mice after water or DSS administration. SMYD5 was not detected in Smyd5ΔIEC mice. Scale bars: 50 μm. ∗∗P < .01, n = 3. (D) Representative IHC staining (left) and quantitative analysis (right) of PGC-1α (brown color) in intestinal epithelia from Smyd5fl/fl and Smyd5ΔIEC mice after water or DSS administration. Scale bars: 50 μm. DSS vs water-treated, ###P < .001; Smyd5ΔIEC vs Smyd5fl/fl, ∗P < .05, ∗∗P < .01; n = 3. (E) HCT116 cells were treated with TNF-α (20 ng/mL) or vehicle control (PBS) for 24 hours, and the expression levels of SMYD5, PGC-1α, and GAPDH were determined by Western blot analysis. The band intensity of SMYD5 and PGC-1α expression in TNF-α–treated HCT116 cells was quantified, normalized to GAPDH, and presented as fold changes relative to the vehicle control-treated cells. ∗P < .05, ∗∗P < .01; n = 3. (F) HCT116 cells were treated with IFN-γ (20 ng/mL) or vehicle control (PBS) for 24 hours, and the expression levels of SMYD5, PGC-1α, and GAPDH were determined by Western blot analysis. The band intensity of SMYD5 and PGC-1α expression in IFN-γ–treated HCT116 cells was quantified, normalized to GAPDH, and presented as fold changes relative to the vehicle control-treated cells. ∗P < .05, ∗∗P < .01; n = 3. (G) Representative Western blots (left) of PGC-1α and GAPDH expression in whole-cell lysates of IECs isolated from Smyd5fl/fl and Smyd5ΔIEC mice. PGC-1α expression (right) was quantified (normalized to GAPDH) and presented as the fold change relative to the level in Smyd5fl/fl IECs. ∗P < .05, n = 3. (H) Pgc-1α mRNA was assessed by RT-qPCR (presented as relative fold change) in IECs isolated from Smyd5fl/fl and Smyd5ΔIEC mice. P = .4346, n = 3.