2.9. Reverse transcription quantitative polymerase chain reaction (RT–qPCR)

TRIzol reagent (Invitrogen) was used to extract total RNA from BMMSCs. With a TaKaRa PrimeScript RT Reagent Kit from TaKaRa, cDNA was produced by reverse transcription of RNA. The reverse‐transcribed cDNA products were diluted with 10 μL of DEPC water. Followed‐up PCR reaction was conducted by a SYBR® Premix Ex TaqTM (Perfect Real Time) kit (TaKaRa). The following settings were used to conduct the reactions: 95°C for 15 s (40 cycles), 60°C for 35 s; 72°C for 30 s; 65°C for 15 s; 95°Cfor 0 s, 0.5°C/s. Table 1 lists the amplification primer sequences.

Primer sequences for real time‐polymerase chain reaction.

F 5′‐GGACCATTCCCACGTCTTCAC‐3′

R 5′‐CCTTGTAGCCAGGCCCATTG‐3′

F 5′‐GCACCCAGCCCATAATAGA‐3′

R 5′‐TTGGAGCAAGGAGAACCC‐3′

OSX

F 5′‐GCCTACTTACCCGTCTGACTTT‐3′

R 5′‐GCCCACTATTGCCAACTGC‐3′

Col‐1

F 5′‐TTCCCGGTGAATTCGGTCTC‐3′

R 5′‐ACCTCGGATTCCAATAGGACCAG‐3′

Fibronectin

F 5′‐CCAGTTAGGGTTGGCGTCTTC‐3′

R 5′‐GCTGGTCCATGCTCAGAGTGTC‐3′

Integrin β1

F 5′‐CCGCGCGGAAAAGATGAATTT‐3′

R 5′‐AGCAAACACACAGCAAACTGA‐3′

Beclin

F 5′‐CAGTACCAGCGGGAGTATAGTGA‐3′

R 5′‐TGTGGAAGGTGGCATTGAAGA‐3′

LC3

F 5′‐CCTGTCCTGGATAAGACCAAGTT‐3′

R 5′‐CTCCTGTTCATAGATGTCAGCGAT‐3′

Fas

F 5′‐TTCCCATCCTCCTGACCAC‐3′

R 5′‐CTCGTAAACCGCTTCCCTC‐3′

FasL

F 5′‐ATTGGCACCATCTTTACTTACC‐3′

R 5′‐CTCCTTAGAATCTGCTCTCATA‐3′

let‐7a

F 5′‐GTGTATCATACAGTATAATGAAACTAC‐3′

R 5′‐AACAGTGCAGTTAGTTCT‐3′

GAPDH

F 5′‐GGCACAGTCAAGGCTGAGAATG‐3′

R 5′‐ATGGTGGTGAAGACGCCAGTA‐3′