Sh nc

Three shRNAs targeting the mouse HSP60 mRNA and a nontargeting control shRNA (shNC) were designed and synthesized by Hanbio Biotechnology (Shanghai, China). Three sequences targeting HSP60 mRNA were as follows: 5′-GATCCGTGCCACTGTTCTGGCACGATCTATTCTCGAGAATAGATCGTGCCAGAACAGTGGCATTTTTTG-3′, 5′-GATCCGATGTTGGCTGTGGATGCTGTAATTCTCGAGAATTACAGCATCCACAGCCAACATCTTTTTTG-3′, and 5′-GATCCGTCAGTCCATTGTCCCTGCTCTTGAACTCGAGTTCAAGAGCAGGGACAATGGACTGATTTTTTG-3′. These sequences were cloned into the vector pHBLV-shRNA-ZsGreen-PURO, and adenoviruses carrying the recombinant vector were generated as previously described (62 (link)).

Small interfering RNAs (siRNAs) targeting the Kdm4e (si-Kdm4e), Dux4 (si-Dux4), and scramble siRNA (si-NC) genes were constructed by Tsingke Biotechnology Co., Ltd. (Beijing, China). Cells grown to 60% confluence were transfected with these siRNAs using Xfect RNA transfection reagent (TaKaRa, 631450) according to the manufacturer’s protocol. The final concentrations of siRNAs was 100 pM. Adenoviruses carrying short hairpin RNAs targeting the Rptor gene (sh-Rptor#1 and sh-Rptor#2) or a scramble RNA (sh-NC) were constructed by Hanbio Co., Ltd. (Shanghai, China). Cells at 60% confluence were transfected with sh-Rptor#1, sh-Rptor#2, or sh-NC for 8 h at an MOI of 50. The medium was removed and fresh medium added, and the cells were then cultured for 48 h before use in experiments. The siRNA and shRNA sequences are listed in supplementary Table 3.

Recombinant lentiviruses for knocking down LINC01003 (sh-LINC01003) and the corresponding control viruses (sh-NC) were purchased from HANBIO (Shanghai, China). Glioma cell U87MG (15 × 10⁴ cells/well) were seeded into a 6-well plate and transfected with the lentivirus according to the manufacturer’s instructions. Infected cells were treated with 5 μg/ml puromycin for more than 7 days.

The lentivirus expressing small hairpin RNA sh-OIP5-AS1 or negative control (sh-NC) was obtained from Hanbio Co., Ltd. (Shanghai, China) and then transduced into NPC cell lines using polybrene (Hanbio Co., Ltd.). miR-203-inhibitor or negative control (GenePharma Co., Ltd., Shanghai, China) was transfected into cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The sequence of sh-OIP5-AS1 is as follows: 5′-CAAACAGGCUUUGUGUUCCUUAUCA-3′.

We used a previously described mice model of unilateral hindlimb ischemia 1 (link). In brief, the mice (male, 12-14 week-old) were anesthetized with a mixture of oxygen and 1.125% isoflurane. Left unilateral femoral artery occlusion was performed by double ligation of the left superficial femoral artery proximal and distal to the deep femoral artery. Animal numbers are stated with the different experimental results. A sham operation was performed on the contralateral right leg. At the same time, for activation or inhibition of SIRT1, the mice were treated with SIRT1 agonist RSV (2 mg/kg/d, J&K) or inhibitor EX527 (2 mg/kg/d, Cayman) by intraperitoneal injection. For intramuscular injection of AAV 21 (link), 3 weeks before SIRT1-Tg mice were performed left unilateral femoral artery occlusion, left gastrocnemius muscles were injected with 1×10¹² vg /mL AAV9-shRNA-cZFP609 (adeno-associated virus-9 short-hairpin RNA; shcZFP609, HANBIO) and AAV9-shRNA-NC (shNC, HANBIO) by multi-point injection, the injection volume was 10 μL per point, 5 points in total. On day 14 after surgery, mice were euthanized by injection of pentobarbital (80 mg/kg IP) 22 (link), the gastrocnemius tissues were harvested and placed in FSC 22 Frozen Section Media (Leica, 3801480), and three random 10 μm frozen sections of the gastrocnemius muscle per animal were used for immunofluorescence analyses.