Fig 2: Effects of FMOD on autophagy and apoptosis in chicken granulosa cells. (A) Relative FMOD mRNA expression levels in normal follicles and atresia follicles. (B) Relative mRNA expression levels of FMOD in ctrl and FMOD silenced cells. (C) Relative mRNA expression levels of PRLR, FSH-β and LH-β in ctrl and FMOD silenced cells. The histogram below shows the gray value analysis of protein. (D) Western blot analysis FMOD protein levels in ctrl and FMOD silenced cells. (E) Relative mRNA expression levels of Belin1, ATG5 and ATG7 in ctrl and FMOD silenced cells. (F) Western blot analysis the expression protein levels of LC3B and Beclin1 in ctrl and FMOD silenced cells. The histogram on the right shows the gray value analysis of protein. (G) Relative mRNA expression levels of caspase-3, caspase-8 and caspase-9 in ctrl and FMOD silenced cells. (H) Apoptosis rates in ctrl and FMOD-silenced cells. (I) The ultrastructure of apoptotic bodies was observed by electron microscopy. (J) Western blot analysis the expression protein levels of caspase3 and capase8 in ctrl and FMOD silenced cells. The histogram below shows the gray value analysis of protein. Date represent mean ± S.E. n = 3 independent cell cultures in each group. *P < 0.05, **P < 0.01.

Fig 3: FMOD regulates the effect of autophagy on apoptosis. (A) Electron microscopy observed the apoptotic bodies in FMOD-silenced cells after treated with Rap, 3-MA, and DMSO, respectively. (B) Flow cytometry detected apoptosis rates of FMOD silenced cells after treated with Rap, 3-MA, and DMSO. (C) Western blot analysis the expression protein levels of caspase3 and capase8 in FMOD-silenced cells after treated with Rap, 3MA and DMSO. The histogram on the right shows the gray value analysis of protein. Date represent mean ± S.E. n = 3 independent cell cultures in each group. Bars not sharing the same letter labels are different (P < 0.05).

Fig 4: FMOD knockdown showed a profound effect on EGFR expression and signaling(A) The differentially expressed genes (DEGs) after FMOD depletion were shown in the volcano plot.(B) The heatmap reveals the DEGs (log2FC > 2, p < 0.05) after FMOD knockdown.(C) RNA-seq analysis revealed the transcripts’ abundance of EGFR after FMOD knockdown.(D) The RNA-seq data was verified using qRT-PCR assay. The EGFR mRNA level was significantly decreased after FMOD knockdown.(E) GO/KEGG analysis based on the RNA-seq data (GSE227643) showed that DEGs by FMOD knockdown were enriched in the EGFR and the MAPK signaling axis.(F) Gene expression correlation analysis was performed. FMOD was highly co-expressed with MAPK signaling pathway.(G and H) GSEA analysis based on the gene highly co-expressed with FMOD showed that FMOD was involved in HNSCC and EGFR signaling. ∗∗, p < 0.01.

Fig 5: FMOD was significantly overexpressed in OSCC(A) Pan-cancer analysis of FMOD expression using TCGA database. The red triangle represents upregulation, while the blue triangle represents downregulation.(B) The FMOD mRNA level (FPKM value) in GTEx normal tissues and TCGA_OSCC tumor tissues.(C) The FMOD mRNA level (FPKM value) in 43 paired OSCC tissues in the TCGA cohort.(D) Immunofluorescence analysis revealed the subcellular location of FMOD protein in OSCC cell lines (SCC-15 and CAL-27) and normal oral cells (HOK-8).(E) WB analysis of FMOD protein in OSCC cell lines (SCC-15 and CAL-27) and normal oral cells (HOK-8). The bar graph shows that FMOD was expressed in CAL-27 and SCC-15 cells.(F) Representative immunohistochemical staining of FMOD in oral normal tissue and OSCC. FMOD was undetectable in 7 samples and very weakly expressed in 8 samples. (a) FMOD staining in normal oral tissues. (b) Weak FMOD staining in #16 OSCC, TNM: I, T1N0M0. (c) Medium FMOD staining in #43 OSCC, TNM: IV, T3N2M0. (d) Strong FMOD staining in #31 OSCC, TNM: IV, T2N2M0.(G) Expression levels of FMOD were higher in OSCC than those of the normal oral tissues. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗p < 0.001.