BMPR2 protein, human

Immunofluorescence staining was performed as described [33 (link), 83 (link), 84 (link)]. Briefly, exponentially growing cells were fixed with 4% paraformaldehyde (PFA) for 10 min, washed with PBS and permeabilized with 1% NP-40 for 10min at room temperature. After being blocked with 10% donkey serum (Jackson Immuno-Research Laboratories, West Grove, PA) for 1h at room temperature, cells were incubated with various primary antibodies, including CD29, CD73, BMPRII, CD90, CD117/c-kit, CD105/endoglin, or BMPR-II antibody (all from Santa Cruz Biotechnology) for 1h at room temperature. Cells were washed with PBS and incubated with FITC-labeled secondary antibodies (Jackson ImmunoResearch Laboratories) for 30 min. DAPI (Invitrogen) was used to visualize nuclei. Stains were examined under a fluorescence microscope. Negative control cells were performed under the same conditions without primary antibodies. Representative images from at least three independent staining experiments are shown.

Total RNA was extracted from freshly isolated human Sertoli cells using Trizol (Takara, Kusatsu, Japan), and the quality and concentrations of total RNA from human Sertoli cells were measured by Nanodrop (Thermo). The ratios of A₂₆₀/A₂₈₀ of total RNA were set as 1.9–2.0 to ensure good purity. In addition, RNA integrity and quality were measured using an Agilent 2100 Bioanalyzer. RNA samples with RNA Integrity Number (RIN) values of more than 7.0 were used for RT-PCR and real-time PCR. Reverse transcription (RT) of total RNA was conducted using the First Strand cDNA Synthesis Kit (Thermo Scientific, USA), and PCR of the cDNA was carried out according to the protocol as described previously49 (link). We detected a number of genes, including WT1 (Wilms tumor1), SOX9 (Sex Determining Region Y-Box 9), BMP4, GDNF, GATA1 (GATA binding protein 1), GATA4, SCF, FGF2, AR, VASA, BMP6, ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B, BMPR2 and ACTB. The primer sequences of these genes were designed and listed in Table S1. The PCR reactions started at 94 °C for 2 min and were performed in terms of the following conditions: denaturation at 94 °C for 30 sec, annealing at 55–60 °C for 45 sec as listed in Table S1, and elongation at 72 °C for 45 sec, for 35 cycles. The samples were incubated for an additional 5 min at 72 °C. PCR with water but without cDNA served as a negative control. PCR products were separated by electrophoresis with 2% agarose gel and they were visualized with ethidium bromide. The band intensities of PCR products were analyzed using chemiluminescence (Chemi-Doc XRS, Bio-Rad).
Quantitative real-time PCR reactions were performed using Power SYBR^® Green PCR Master Mix (Applied Biosystems, Woolston Warrington, UK) and a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). To quantify the PCR products, the comparative Ct (threshold cycle) method was used as described previously50 (link). The threshold of cycle values of genes was normalized against the threshold value of human housekeeping gene ACTB [ΔC_T = C_T( gene)− C_T(ACTB)], and the relative expression of genes in treated group to the control was calculated by formula 2^−ΔΔCT [ΔΔC_T = ΔC_T(treated)−ΔC_{T (control)}]. The primer pairs of detected genes were listed in Table S1. The Ct values and a series of five-fold dilutions of template cDNA of human Sertoli cells were utilized for drawing the standard curves, and the slope of each standard curve was used to calculate the efficiency (E) of gene primers using the formulae: E = 10^(−1/slope)-1, according to the method described previously51 (link)52 (link)53 (link)54 (link).

qRT-PCR was performed as previously described [27 (link)]. Total RNA was isolated using ISOSPIN Cell & Tissue RNA (Nippon Gene, Tokyo, Japan). Then, 100 ng of total RNA was transcribed to cDNA using the TaqMan Universal Master Mix. Mouse beta-actin (4352341E, Applied Biosystems, Foster City, CA, USA) was used as the endogenous control. qPCR was performed using a QuantStudio 12K Flex instrument (Applied Biosystems). The primers and probes were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The assay identification of each primer and probe are as follows: bone morphogenetic protein receptor type-2 (BMPR2; Mm03023976_m1); transforming growth factor-b1 (Tgf-b1; Mm 01178820_m1); plasminogen activator inhibitor-1 (PAI-1; Mm00435860_m1); interleukin-6 (IL-6; Mm00446190_m1); and tumor necrosis factor (TNF; Mm00443258_m1).

Total protein was extracted from cells with RIPA lysis buffer (NCM Biotech, China), and the protein concentration was determined with a BSA protein detection kit (Beyotime, China). Proteins were separated on a 12% SDS‒PAGE gel and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies (Abcam, USA) overnight at 4 °C, including Runt-related transcription factor 2 (Runx2, ab236639, dilution ratio 1:1000), NF-κB-inducing kinase (NIK, ab203568, dilution ratio 1:500), p65 (ab32536, dilution ratio 1:1000), P-p65 (ab76302, dilution ratio 1:1000), BMP receptor type 2 (BMPR2, ab130206, dilution ratio 1:1000), Smad2/3 (ab202445, dilution ratio 1:1000), P-smad2/3 (ab254407, dilution ratio 1:1000), Smad4 (ab230815, dilution ratio 1:1000), iNOS (ab283655, dilution ratio 1:1000), arginine (Arg, ab203490, dilution ratio 1:1000), type I collagen (Col-I, ab138492, dilution ratio 1:5000), c-fos (ab222699, dilution ratio 1:1000), TRAP (ab52750, dilution ratio 1:5000), p38 (ab170099, dilution ratio 1:1000), P-p38 (ab4822, dilution ratio 1:1000), NADPH oxidase 2 (NOX2, ab129068, dilution ratio 1:1000), glutathione peroxidase (GPX4, ab125066, dilution ratio 1:1000), SOD2 (ab68155, dilution ratio 1:1000) and β-actin (ab8226, dilution ratio 1:1000). After being washed, the membranes were incubated with the corresponding secondary antibodies (goat anti-mouse, ab205719, goat anti-rabbit, ab205718, dilution ratio 1:10,000) for 2 hours. The target proteins were visualized using ECL reagents (Thermo Fisher Scientific, USA), and digital images were taken using a ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, USA). All blots were processed in parallel and derive from the same experiment. Band intensity was quantified using ImageJ (National Institutes of Health, USA).

Total proteins were extracted from tissues and cells using RIPA buffer (Cell Signaling). Equivalent amounts of protein determined by BCA assay were resolved by 10% Bis–Tris gel electrophoresis and blotted to Immobilon PVDF membranes (Millipore). The following primary antibodies and dilutions were used: anti-BMP4 (1:1000; ab39973, Abcam); anti-GAPDH (1:2000; sc-32233, Santa Cruz);anti-Flag (1:1000; F1804, Sigma); anti-ACVRIIA (1:1000; ab134082, Abcam); anti-ACVRI (1:1000; 4398 s, Cell Signaling); anti-BMPRII (1:1000; 6979 s, Cell Signaling); anti-p-Smad1/5/8 (1:1000; 13820 s, Cell Signaling); anti-Smad1 (1:1000; 9743 s, Cell Signaling); anti-P85 (1:1000; 4292S, Cell Signaling); anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:1000; 4668S, Cell Signaling); anti-SAPK/JNK (1:1000; 9252 s, Cell Signaling); anti-P-ERK1/2 (1:1000; 8544S, Cell Signaling); anti-ERK1/2 (1:1000; 4695S, Cell Signaling); anti-P-P38 (1:1000; 4511S, Cell Signaling); anti-P38 (1:1000; 8690S, Cell Signaling); anti-Capspase3 (1:1000; 9662S, Cell Signaling); anti-LC3B (1:1000; ab192890, Abcam); anti-Parkin (1:1000; ab77924, Abcam); anti-P62 (1:1000; ab109012, Abcam); anti-PGC1 alpha + beta (1:1000; ab188102, Abcam). The secondary antibody was horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Jackson). The ratio of band intensity of target protein to that of GAPDH was calculated as the relative expression level of protein.