Lipo 6000 transfection reagents

HEK293T cells were transfected with pLKO.1 plasmids with targeted shRNA sequences for gene knockdown or pLJM1 with an Arg2 gene insert for gene overexpression together with psPAX2 and pMD2.G plasmids using Lipo 6000 Transfection Reagents (Beyotime Biotechnology) to generate lentivirus particles. After transfection, HEK293T cells were cultured for 48 h, and then the supernatant was centrifuged at 1500
g for 5 min to collect viral particles. The sequences of the sense strand targeting mouse GPX4 and Arg2 are as follows:
GPX4 shRNA: 5′-GTCGATCTGCATGCCCGATAT-3′;
Arg2 shRNA: 5′-CACAAGATGATCCCTACAATA-3′; and scramble shRNA (5′-CCTAAGGTTAAGTCGCCCTCG-3′) was a gift from David Sabatini’s Lab deposited in Addgene (Cambridge, Watertown, USA)
[29] (link).

hBMSCs were purchased from Cyagen Biosciences (Guangzhou, China). hBMSC growth medium were purchased from Cyagen Biosciences. Cells cultured in a atmosphere of 5% CO₂ at 37 °C. Osteogenic induction medium was prepared according to previous methods [49 (link)].
SERPINB2 recombinant protein was purchased from Amyjet Scientific (Abnova, wuhan, china). Antibodies used for western blotting including GAPDH (1:1000, Beyotime, shanghai, china), RUNX2 (1:1000, Beyotime), COL1A1 (1:1000, Beyotime), SERPINB2 (1:1000, Abcam), SP7/Osterix (1:1000, Abcam, shanghai, china), non-phosphorylated (active) β-catenin (1:1000; Cell Signaling Technology), or total β-catenin (1: 1000; Beyotime), phosphorylated and total ERK (1:1000; Cell Signaling Technology), phosphorylated and total AKT (1:1000; Cell Signaling Technology). Second antibodies Alexa Fluor 555 was purchased from Beyotime. Lipo6000 Transfection Reagents were purchased from Beyotime (Shanghai, china).
The siRNAs were purchased from Genepharma (Hangzhou, china).
hSERPINB2 sense: GCAAAGAAUCAAGUUGCAATT
hSERPINB2 antisense: UUGCAACUUGAUUCUUUGCTT
hNC sense: UUCUCCGAACGUGUCACGUTT
hNC antisense: TTAAGAGGCUUGCACAGUGCA
mSERPINB2 sense: GGAGAGAAGUCUGCAAGAUTT
mSERPINB2 antisense: AUCUUGCAGACUUCUCUCCTT
mNC sense: GGCUCUGTTCUUGUCACGUAA
mNC antisense: UUTTGAGGCAAGCACAGUGCA