Flx800t

Following siRNA-Scr, Rg3 or/and siRNA treatment for 72 h, GBC-SD cells were incubated with RPMI-1640 medium containing 10 µM 2′,7′-dichlorofluorescein (DCF) diacetate for 1 h at 37°C, following termination of the reaction with 0.25% trypsin and washed twice with PBS. The intensity of DCF fluorescence was quantified using a fluorescence analyzer (FLX800T; BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 408 nm. The cells were transfected with siRNA-Scr (50 nM) as negative control. The experiments were performed in triplicate.

To quantify the myogenic gene expressions of hASCs, markers Myog, Myh4, Myh5 myogenic markers were analyzed via RT-PCR after 14 d of culture. Briefly, the total RNA of the hASCs from CC- and ISC- construct were isolated using a Tri reagent (Millipore Sigma). To measure the concentration and purity of the isolated RNA, a spectrophotometer (FLX800T; Biotek, USA) was used. Then, complementary DNA (cDNA) was synthesized from the RNase-free DNase-treated total RNA using a ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd., Japan). The synthesized cDNA was used to conduct quantitative RT-PCR. StepOnePlus real-time PCR system (Applied Biosystems, USA) was used to conduct RT-PCR and the expression of the myogenic gene was normalized using expression levels of beta actin (Actb). In addition, gene levels of hASCs on CC- structure were set as one-fold. The gene-specific primers used are as follows: human Myog (NM_002479.6; left: 5′ - gct cag ctc cct caa cca −3′, right: 5′ - gct gtg aga gct gca ttc g − 3′), human Actb (NM_001101.5; left: 5′ - tcc aaa tat gag atg cgt tgt t − 3′, right: 5′ - tgc tat cac ctc ccc tgt gt − 3′), human Myh4 (NM_017533.2; left: 5′ - tgc tgg ctt tga gat ctt tg − 3′, right: 5′ - tgc agt ttc tcg ttg gtg aa − 3′), and human Myf5 (NM_005593.3; left: 5′ - cta tag cct gcc ggg aca −3′, right: 5′ - tgg acc aga cag gac tgt tac at − 3′).