Chrm1

Quantitative RT-PCR (qRT-PCR) was done as reported 12 (link). Primers for GAPDH, HHIP, PTCH1, CD31, GLI1, AQP5, BDNF, NRTN, C-KIT, P21, AMPK, ULK1, Chrm1, CHRM3 and VEGFA were designed with the Primer3 software (http://bioinfo.ut.ee/primer3/). For Western blot, fresh parotid samples were homogenized with 40 μL T-PRE reagent containing protease inhibitors (Pierce, USA) per mg followed by centrifugation at 10,000 ×g for 5 min to collect supernatant. Western blot was done as reported 12 (link) with antibodies against CD31 (1:1000, Cell Signaling Technology), LC3B (1:2000, Abcam), P62 (1:5000, Abcam), Chrm1 (1:1000, Abcam) (Details in supplemental methods).

PC-3 cells were seeded in 6-well or 12-well plates and left untreated (control) or treated with carbachol (2 μg ml⁻¹) or pirenzepine (110 μg ml⁻¹) for 2, 4, 6, 12, and 24 h. Cells were lysed in NP 40 buffer (Solarbio, Beijing, China) containing protease inhibitors. Total protein was measured using the Bicinchoninic acid (BCA) method (Beyotime). Primary antibodies against SHH (1:2000, Abcam, Cambridge, MA, USA), PTCH1 (1:1000, Abcam), GLI1 (1:1000, CST, Beverly, MA, USA), and CHRM1 (1:1000, Abcam) as well as horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSbio, Beijing, China) were applied. Equal protein sample loading was monitored using an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5000, Ray Antibody, Beijing, China). The probed proteins were visualized with an enhanced chemiluminescence (ECL) blotting detection kit (BIO-RAD, Chengdu, China).