Cdna synthesis kit

The whole RNA was extracted by using TRIzol kit (Invitrogen, USA) and reversely transcribed into cDNA by using the cDNA synthesis kit (Beyotime, China). SYBR Green qPCR mix (Beyotime, China) was applied to measure the relative expression of Rab8A in the ABI 7900 system. The expression of Rab8A was calculated by using the 2^-ΔΔCt method. Three independent experiments were carried out to determine Rab8A expression. GAPDH was regarded as the internal reference. The sequences of primers were listed as follows: Rab8A, forward: 5′-CTACGACATCACCAACGAGAAG-3′, Rab8A, reverse: 5′-CATCACACTTGTTCCCGAGTAT-3′; GAPDH, forward: 5′-GCACCGTCAAGGCTGAGAAC-3′, GAPDH, reverse: 5′-TGGTGAAGACGCCAGTGGA-3′.

The RNA was extracted by TRIzol reagent (ThermoFisher Scientific, USA) from SW480 cells with different concentrations of TalaA treatment. The cDNA was synthesized with a cDNA synthesis kit (D7170M, Beyotime, China), and the real-time PCR was performed using a SYBR Green qPCR kit (D7260, Beyotime, China) in a light cycler 480 II (Roche, USA). The primer sequences for real-time PCR are listed in Table 1.Table 1

The primer list for real-time PCR.

Name	q-PCR primer
FTL	Forward: 5′-GCTCCCAGATTCGTCAGAA-3′ Reverse: 5′-CCCAGAGAGAGGTAGATGTAGG-3′
FTH1P23	Forward: 5′-CAGGTGAAAGCCATCAAAGA-3′ Reverse: 5′-TTATCACTGTCTCCCAGCG-3′
SAT2	Forward: 5′-TGATTCGGGTGAAGACTGC-3′ Reverse: 5′-TCTCCAAAGCCATCTGCTC-3′
HMOX1	Forward: 5′-ATTGCCAGTGCCACCAAGT-3′ Reverse: 5′-TGAGCAGGAACGCAGTCTT-3′
ALOXE3	Forward: 5′-TGTATTTCGCTTTCCTGACC-3′ Reverse: 5′-CTTGTTTGCTTGCCTCTGA-3′
ACSL5	Forward: 5′-TAGAAGCACTGAGAGATGCG-3′ Reverse: 5′-TTGTGAACAGCAGCAGGA-3′
SLC7A11	Forward: 5′-TATCCCTGGCATTTGGAC-3′ Reverse: 5′-TCACTACAGTTATGCCCACAG-3′
GSS	Forward: 5′-ACAGGATGACTTTACCGCTC-3′ Reverse: 5′-AGCGGTAAAGTCATCCTGTT-3′

Total RNA from cells was purified using an RNeasy kit (Beyotime Biotechnology, R0026), and reverse transcribed into cDNA using a cDNA synthesis kit (Beyotime Biotechnology, D7170M). The Egr-1 primer sequences were as follows: forward, 5’-CCGCTTTTCTCGCTCGGATG-3’; reverse, 5’-GCGGATGTGGGTGGTAAGGT-3’. The LC3A primer sequences were as follows: forward, 5’-CGTCACCCAGGCGAGTTACC-3’; reverse, 5’- AGAGATGCGTCTGCGGTTCG-3’. The LC3B primer sequences were as follows: forward, 5’- GTGATCGTCGCCGGAGTCAG-3’; reverse, 5’-CGCTCTATAATCACTGGGATCTTGG-3’. The NRF2 primer sequences were as follows: forward, 5’-GTCGCCGCCCAGAACTGTAG-3’; reverse, 5’-AAGGTGCTGAGCCGCCTTTT-3’. The MACRO primer sequences were as follows: forward, 5’-TGGGCACCCAAAACACACCT-3’; reverse, 5’-TGGACCTGGAGAGCCTCGTT-3’; The MSR1 primer sequences were as follows: forward, 5’-CTGGGCAGAGCACCCTACTATC-3’; reverse, 5’-AAAAGGTGCCAGGGGATGGGA-3’. The primers were designed by Primer-BLAST (NCBI). RT-qPCR assays were conducted in triplicate using the SYBR green method on QuantStudio 5 Real-Time PCR System (Applied Biosystems) according to manufacturer’s instructions. β-actin was used as housekeeping control mRNA. Data were analyzed using the relative standard curve method according to the manufacturer’s protocol.

Ginsenoside Rg1 (>98%, Aladdin, China); Gelatin (Porcine skin, Type A, Sigma-Aldrich, United States); Span 80 (Aladdin, China); Olive oil (Aladdin, China); SrCl₂⋅6H₂O (Aladdin, China); Gibco MEM α, Nucleosides (Thermo Fisher Scientific, United States); Fetal bovine serum (FBS, Sigma-Aldrich, United States); Anti-Osteocalcin (Anti-OCN, Santa Cruz Biotechnology, United States); CCK-8 Kit (Beyotime, China); ALP Kit (Beyotime, China); RNeasy Mini Kit (Qiagen, United States); cDNA synthesis Kit (Beyotime, China); Bulge-LoopTM miRNA qRT-PCR kit (Ribobio, Guangzhou, China); Hematoxylin and Eosin Staining Kit (Beyotime, China); Masson’s Trichrome Stain Kit (MKbio, Shanghai, China); and Safranin O-Fast green Stain Kit (Servicebio, Wuhan, China).