Gb11009 1

Freshly dissected penile and MPG tissues were harvested and fixed with 4% paraformaldehyde for further histological staining analysis. Masson trichrome staining was first carried out to determine smooth muscle and collagen expression levels in penile tissues. A 5‐μm slice was prepared and stained following the manufacturer's instructions, and the smooth muscle was stained red while the connective tissue appeared blue. For immunofluorescence staining, the penile sections were incubated with primary antibodies including α‐SMA (1:300, Servicebio, GB111364), eNOS (1:500, Abcam, ab66127), nNOS (1:500, Servicebio, GB11145), Caspase‐3 (1:200, Servicebio, GB11009‐1), and the MPG sections were covered by primary antibodies including Tuj1 (1:2500, Servicebio, GB11139), and GFAP (1:2500, Servicebio, GB11096) at 4°C overnight. After rinsing the slices with PBS, secondary antibodies were adopted for 1‐h immersion. Nuclei were stained with DAPI. Images were observed, and the interesting areas were captured under a confocal laser scanning microscope (Zeiss LSM 710) and a fluorescence microscope (NIKON, Ti2). FIJI/ImageJ software (National Institutes of Health) was used to carry out image analysis.

The immunohistochemistry in this experiment included the following antibodies: caspase-3 (CASP3; GB11009-1; Servicebio, Wuhan, China; 9 rats from each of the CA and CM groups); interleukin-6 (IL-6; GB11117; Servicebio; 13 and 14 rats from the CA group and CM groups, respectively); mammalian target of rapamycin (mTOR; GB11117; Servicebio; 13 rats from each of the CA and CM groups); nuclear factor-kappa B (NF-κB; 13,533–1- AP; Proteintech, Wuhan, China; 9 rats from each of the CA and CM groups); and tumor necrosis factor-alpha (TNF-α; bsm-33207 m; Bioss, Beijing, China; 13 and 14 rats from the CA group and CM groups, respectively). The experimental method of this part is in the Additional file 3: Experimental methods.

The pathway/ontology enrichment analysis of mutations genes was performed using the Metascape (http://metascape.org). The statistical tests were conducted at a two-sided level of significance of 0.05 in R environment. Immunohistochemical analysis for Ki67 and cleaved-caspase-3 was performed using paraffin sections. Immunohistochemical staining was carried out for cleaved-caspase-3 (1:200; Servicebio, GB11009-1) and Ki67 (1:600; Servicebio, GB121141).