Rneasy plus mini kit

Total RNA was used to extract by RNeasy plus mini kits (Tiangen). Real-time PCR was showed as previously described [18 (link)]. 36B4 was used for internal reference. The primer sequences were displayed here. RNF181: F: cac aga cga gat aag gct cga a; R: tgg cca ggt ctg tga tca at. 36B4: F: ggc gac ctg gaa gtc caa ct; R: cca tca gca cca cag cct tc. CTGF: F: ctc gcg gct tac cga ctg; R: ggc tct gct tct cta gcc tg. CYR61: F: agc agc ctg aaa aag ggc aa; R: agc ctg tag aag gga aac gc.

hPDLCs were seeded in 6-well plates at a density of 3 × 10⁵ cells/well and treated with hypoxia condition (1% O₂) or succinate (0, 1, and 5 mmol/L) for 4 h. The total RNA was extracted with the RNeasy Plus Mini Kit (Tiangen, China). Reverse transcription reaction was performed with 1 μg of RNA using a cDNA synthesis kit (Vazyme, China). Then, the cDNA was mixed with the primers (GenScript, China) and Maxima® SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) according to the manufacturer's protocol and subjected to real-time PCR on the ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific, USA). β-Actin was used as the internal reference gene, and data were analyzed with the 2^−ΔΔCt method. Results were shown as the relative expression ratio of the target gene to the reference gene. The sequence of primers is listed in Table 1.

Total RNA was extracted with an RNeasy Plus Mini Kit (Tiangen, DP451) following the manufacturer’s specifications. Reverse transcription was performed using HiScript II Q RT SuperMix (Vazyme, R223-01). qRT–PCR was carried out using SYBR qPCR Master Mix (Vazyme, Q511-02) and a 7500 Fast Real-Time PCR System (Applied Biosystems, Singapore). 36B4 was used as an internal control. The sequences of the primers used for qPCR were as follows: 36B4 F: GGC GAC CTG GAA GTC CAA CT; R: CCA TCA GCA CCA CAG CCT TC. CTGF F: CTC GCG GCT TAC CGA CTG; R: GGC TCT GCT TCT CTA GCC TG. CYR61 F: AGC AGC CTG AAA AAG GGC AA; R: AGC CTG TAG AAG GGA AAC GC. DUB1 F: GAG GCC GGG GCT CTG A; R: ACT GGG ATG TGC AGA CTT GG. TAZ F: AGA GTC GGG TCG GGA TTT GT; R: AGG CCG GAT TCA TCT TCT GGG.