Article Title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner
Figure Lengend Snippet: Inhibitors of v-ATPase are the most potent activators of MTORC1 in chondrocytes. ( A ) MEFs and C5.18 chondrocytes were treated with varying doses of concanamycin for 48 h. ( B ) C5.18 chondrocytes were treated with varying doses of Baf, 3-MA, CQ and leupeptin for 48 h. In all panels the levels of p-RPS6 and SQSTM1 were analyzed by western blot.
Article Snippet: Materials and Methods The following chemicals and reagents were used in the preparation of this manuscript: Phosphate buffered saline (PBS; Invitrogen, 1160381), Dulbecco's modified Eagle's medium/F-12 mixture (DMEM/F12; Invitrogen, 11330057), DMEM custom medium (made by Life Technologies and is a standard DMEM medium depleted of all amino acids, glucose and pyruvate), minimal essential medium (Invitrogen, 22571020), fetal bovine serum (FBS; Life Technologies, 16000-044), gentamycin (Invitrogen, 15750), ascorbic acid (Sigma-Aldrich, A5960), β-glycerophosphate (βGP; Sigma-Aldrich, G9422), doxycycline (dox; Sigma-Aldrich, D3447), bafilomycin A1 (LC laboratories, B-1080), concanamycin A (Sigma-Aldrich, 27689), chloroquine (Sigma-Aldrich, C6628), dexamethasone (Sigma-Aldrich, D1756), Torin1 (Tocris Biosciences, 4247), hyaluronidase (Sigma-Aldrich, H3506), leupeptin (VWR, a2183.0025), ECL western blotting substrate (Perkin Elmer, NEL104001EA), 2x Laemmli buffer (Sigma-Aldrich, S3401), alcian blue (Sigma-Aldrich, A5268), digoxigenin (Roche Inc., 11277073910), bovine serum albumin (Sigma-Aldrich, A8806), IGF1 (Sigma-Aldrich, I3769), Magic Cathepsin B kit (ImmunoChemistry Technologies, 937), rat-tail collagen (Invitrogen, A1048301), microscope cover glasses (VWR International, ECN 631-1578), 3-methyladenine (Sigma-Aldrich, M9281), Vectastatin avidin-biotin complex (ABC) kit (Vector laboratories, PK-6100), 3, 3′-diaminobenzidine kit (DAKO, K3468), and normal horse serum (DAKO, S-2000).
Techniques: Western Blot