Fig 2: Celecoxib does not affect COX RNA transcript expression in myoblasts. (a) Myoblasts were treated with various concentrations of Celecoxib (a), NS-398 (b), or vehicle (DMSO) in growth media for 48 h. Cells were harvested 48-h after seeding, RNA was extracted, and RT-PCR performed using primers/probes for PTGS1 (COX-1) or PTGS2 (COX-2) and normalized to GAPDH gene expression. Data are expressed as mean ± SD from four independent experiments, each performed in triplicate.

Fig 3: EMPA treatment increased mitochondrial biogenesis. (A) Left, Transmission electron microscopy showing the morphology of mitochondria in sham, sham + EMPA, TAC and TAC + EMPA groups and Right, quantitative analysis of mitochondrial size and counts. Results are expressed as mean ± SEM, n = 3–5, *p < 0.05 vs. corresponding sham, †p < 0.05 vs. corresponding TAC. (B) Left, Representative blots of mitochondrial biogenesis-related proteins and Right, quantitative results. (C) Relative mRNA levels of PGC1a, NRF1, TFAM and COX1. Results are expressed as mean ± SEM, n = 5–7, *p < 0.05 vs. corresponding sham group, †p < 0.05 vs. corresponding TAC vehicle group. One-way ANOVA and Tukey post hoc test. EMPA, empagliflozin; SEM, standard error of the mean; TAC, transverse aortic constriction; PGC1-a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; NRF-1, nuclear respiratory factor 1; TFAM, mitochondrial transcription factor A; COX1, cyclooxygenase1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig 4: Celecoxib alters COX mRNA, but not protein, in cultured human myotubes. (a) Myoblasts were seeded directly into differentiation media containing the indicated concentrations of celecoxib or diluent control (DMSO). Cells were harvested 72 h after seeding, when differentiation into myotubes was complete. (a) RT-PCR was performed using primers/probes for PTGS1 (COX-1) or PTGS2 (COX-2), first normalizing to GAPDH gene expression, and then normalizing to DMSO control, which was set to 1.0. Data are expressed as mean ± SD and represent three independent experiments (p < 0.001; ** p < 0.01). (b) Western blotting was performed on lysates derived from myotubes exposed to the indicated concentrations of celecoxib. Purified recombinant COX-1 or COX-2 peptides were electrophoresed alongside experimental samples to verify relative molecular masses of COX enzymes. Data from four independent experiments were quantified as described in Figure legend for Figure 10b, and are expressed as mean ± SD (*** p < 0.001). (c) Western blotting was performed on lysates derived from myotubes exposed to NS-398 (or DMSO) for 72 h. No differences in levels of COX enzymes were observed at any concentration.

Fig 5: In vivo tumor growth assessment and RNA-seq characterization of murine BrafV600E melanoma cells with ptgs1, ptgs2, or ptges KO. A, Cell proliferation assay in scramble, ptgs1-KO, ptgs2-KO, and ptges-KO murine BrafV600E melanoma cells. Results represent the fold change relative to the OD value of baseline levels at time 0 for each cell (0, 24, 48, and 72 hours after culture; n = 6). Differences between two groups were analyzed by repeated measures one-way ANOVA, followed by Tukey post hoc test. B, Left, Tumor volume measurement at indicated timepoints for scramble, ptgs1-KO, ptgs2-KO, and ptges-KO cells. A total of 1 × 106 cells per mouse were injected subcutaneously into the flanks of C57BL/6J mice: n = 8 mice each for scramble, ptgs1-KO, and ptgs2-KO groups, and n = 7 for ptges-KO group. Differences in tumor volume between two groups were analyzed by repeated measures one-way ANOVA, followed by Tukey post hoc test. B, Right, Tumor volumes for individual mice in each group are shown. C, Tumor-free survival (tumor volume < 1,000 mm3) was estimated using the Kaplan–Meier method and compared using the log-rank test. D, Heat map showing the unsupervised hierarchical clustering of the mRNA levels (Reads Per Kilobase Million [RPKM]) of eight collagen-related genes across scramble, ptgs1-KO, ptgs2-KO, and ptges-KO cells. RNA was extracted from three different subclones in each cell line. E, Spearman correlation values between immune-suppressive markers and collagen-related genes in melanoma samples from TCGA-SKCM dataset. Graph values represent mean ± SD. **, P < 0.01. N.S., not statistically significant.