Figure 3.

Both HVEM and LTβR are required for myoblast differentiation and survival. (a) HVEM or LTβR mRNA transcripts were measured over the course of C2C12 differentiation by qRT-PCR (n=4 for HVEM and n=3 for LTβR). (b) HVEM and LTβR protein levels during C2C12 cell differentiation were analyzed by western blotting (n=4). (c) C2C12 cells were infected with shRNA lentiviruses as indicated, selected for 2 days, followed by differentiation for 72 h and subsequent staining for MHC and with DAPI. Scale bar: 50 μm. (d and e) Fusion index and total nuclei number were quantified for experiments in (c) (n=5). (f) Lentivirus-mediated knockdown of HVEM and LTβR in C2C12 cells were confirmed by western analysis (n=3 for HVEM and n=4 for LTβR). (g) C2C12 cells treated as in (c) but differentiated for 24 h were subjected to TUNEL assays, and the percentage of apoptotic cells was measured (n=4). One-sample two-tailed t-test was performed for data in (a) and relative nuclei number in (d) and (e), paired two-tailed t-tests for all other data. *P<0.05; **P<0.01. All error bars represent S.D. of independent replicates