Basic fibroblast growth factor (bfgf)

Isolated muscle fibers with primary SCs were cultured in DMEM (high glucose, sodium pyruvate, and GlutaMAX supplement; Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (FBS), 1% chick embryo extract (CEE) (US Biological), and 1% penicillin-streptomycin (PS) (Thermo Fisher Scientific) at 37 °C with 5% CO2. Established iSkM progenitor cells were cultured in DMEM (high glucose, sodium pyruvate, and GlutaMAX supplement) supplemented with 10% FBS, 1% CEE, 5 ng/ml basic fibroblast growth factor (Cell Signaling), 5 ng/ml LIF (PROSPEC), 100 ng/ml dox (LCK Laboratories), and 1% PS. All plastic dishes used for cSCs and iSkM progenitor cells were coated with Matrigel. C2C12, 3T3-L1, MEFs, TTFs, Plat-E, and HEK293T cells were cultured in DMEM (high glucose; Wako) supplemented with 10% FBS and 1% PS at 37 °C with 5% CO2. The medium was changed every 2 days.

ReN VM neural progenitor (catalog number SCC008) and Neuro-2a mouse neuroblast (catalog number CCL-131) cells were obtained from Millipore and ATCC (VA, USA), respectively. The undifferentiated ReN VM cells were grown on Matrigel basement membrane matrix (catalog number 354230, Corning, NY, USA) and maintained in DMEM/F12 (catalog number 11320033, Thermo Fisher Scientific) supplemented with 20ng/ml basic fibroblast growth factor (catalog number 8910, Cell Signaling, MA, USA), 20ng/ml epidermal growth factor (catalog number RKP01133, Reprokine, FL, USA), 10 Units/ml heparin sodium salt (catalog number H3149, Sigma-Aldrich) and 1X N21-MAX (catalog number AR008, R&D Systems, MN, USA) or 1X B27 media supplement (catalog number 17504044, Thermo Fisher Scientific). The cells were differentiated into neuronal and glial populations with the removal of heparin and growth factors from media for at least 7 days. Passage-matched N2a and RML-infected N2a cells were created as described previously [64 (link)] and maintained in DMEM (catalog number 11995065, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (catalog number 12483020, Thermo Fisher Scientific) and 1% GlutaMAX (catalog number 35050061, Thermo Fisher Scientific). CRISPR-Cas9-generated N2a knockout cells were described previously [65 (link)].

Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria Escherichia coli (E. coli) were purchased from Guangdong Microbial Culture Collection (China). Dulbecco's modified Eagle's medium (DMEM), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and fetal bovine serum (FBS) were purchased from Gibco (USA). Human skin keratinocytes cells (HaCat) and human umbilical vein endothelial cells (HUVEC) were obtained from American type culture collection (ATCC). The 2,2’-bis(anthracene-9,10-diylbis(methylene))-dimalonic acid (ABDA) was supplied from Shanghai Civi Chemical Technology Co., Ltd. 1-methyl-2-pyrrolidone (NMP) was purchased from Aladdin. The primary antibodies to CD31, VEGF, bFGF, Tubulin were obtained from Cell Signaling Technology (USA). We used deionized water throughout the experiment.

HeLa S3 cells were maintained in EMEM (Lonza), Primary Fibroblasts were maintained in Quantum 333 media (PAA) or FibroPlus 333 (Capricorn-Scientific) and EBV-transformed lymphoblasts were maintained in RPMI 1640 media (Gibco). All media was supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin. Quantum 333 and FibroPlus 333 media was also supplemented with 10 ng/ml bFGF (Cell Signalling Technology). Cell lines were maintained at 37°C with 5% CO₂ in a humidified incubator.
Fibroblast cell lines were electroporated using the Neon Transfection system (Life Technologies) as per the manufacturer’s instructions using a single pulse of 20 ms and 1600 V. Medium GC content control siRNA and BOD1 Stealth siRNA (5’-GCCACAAAUAGAACGAGCAAUUCAU-3’) were supplied by Life Technologies. The specificity of these reagents and absence of off-target effects were reported previously [16 (link),17 (link)]. Briefly, BOD1 Stealth siRNA does not target Bod1L or Bod1L2 mRNA and all phenotypes associated with the BOD1 Stealth siRNA can be rescued using exogenous siRNA-resistant BOD1 expression constructs.
Unless otherwise indicated RO3306 was used at 9 μM, Monastrol at 100 nM and BI 2536 at 12.5 to 200 nM.

Before starting the induction of Prg4-positive cells, iPSCs were cultured for 4 days in iPSC maintenance medium in a humidified atmosphere with 5% CO₂ at 37 ℃, until they became subconfluent. Initially, iPSCs were differentiated as embryoid bodies on 96-well plates (Nunclon Sphera; Thermo Scientific) in standard high-glucose DMEM containing 10% foetal bovine serum, penicillin, and streptomycin for 2 days. On day 2, the medium was changed to standard medium containing 9 ng/mL activin A (Peprotech) and 25 ng/mL Wnt3a (R&D) for mesoderm differentiation. On day 3, the medium was changed to standard medium containing 10 ng/mL basic fibroblast growth factor (bFGF; Wako), and the cells were cultured continuously for 2 days. On day 5, embryoid bodies were dissociated and reseeded as a monolayer on collagen-coated 6-well plates, containing standard medium supplemented with 1% insulin-transferring-selenium (ITS; Gibco), 10 ng/mL transforming growth factor beta 1 (TGF-β1; Cell Signaling), and 10 ng/mL bFGF. Medium was changed every other day and differentiated cells were cultured until day 21.

The collected cavernous nerve and penis samples were fixed in 4% paraformaldehyde for 24 h at 4 °C before creating a paraffin block. The following primary antibodies were used: Neuronal nitric oxide synthase (nNOS, diluted 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), alpha smooth muscle actin (α-SMA, diluted 1:500; Abcam, Cambridge, UK), vascular endothelial growth factor (VEGF; diluted 1:200; Santa Cruz Biotechnologies), basic fibroblast growth factor (bFGF, diluted 1:500; Cell Signaling Technology), stromal cell-derived factor-1 (SDF-1 diluted 1:200; Abcam), and platelet endothelial cell adhesion molecule (PECAM-1, diluted 1:500; Abcam), and 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA) was used to stain nuclei. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2012 (Zeiss).