Specific primers

After cells had been washed with PBS, total RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific) and complementary DNA (cDNA) was synthesized using a SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific). Real-time RT-PCR was carried out using specific primers (TaKaRa Bio) with a QuantStudio 3 system (Thermo Fisher Scientific). The fluorescence of the PCR products was monitored in real-time using TB Green Premix Ex Taq (TaKaRa Bio). Expression was calculating using the 2^−(Ct−Cc) method relative to that of the control, GAPDH.

Passage 4 BMSCs from SLE patients were cultured in a 6-well plate (2.0 × 10⁵ viable cells). Total cellular RNA was extracted using Trizol reagent (Invitrogen, USA) and stored at −70°C. For quantitative RT-PCR, single-stranded cDNA was synthesized in a 200 μL reaction volume containing 30 μL total RNA using PrimerScript RT reagent Kit (TaKaRa, China). Quantitative RT-PCR was performed on an ABI 7500 FAST real-time PCR detection system (Applied Biosystems, USA) using SYBR Green detection mix (Takara, China). StepOne Software v2.1 was used to detect the mix, and the thermal profile for RT-PCR was 95°C for 30 seconds, followed by 40 cycles of 5 seconds at 95°C with 34 seconds at 60°C. The specific primers (TaKaRa, China) used are shown in Table 2.
Relative expression of the target genes was calculated with the 2^−ΔΔCt method. Briefly, a value for the cycle threshold (Ct) was determined for each sample and defined as the mean cycle at which the fluorescence curve reached an arbitrary threshold. The ΔCt for each sample was then calculated according to the formula Ct target gene − Ct GAPDH; ΔΔCt values were then obtained by subtracting the ΔCt of a reference sample (average ΔCt of the control group) from the ΔCt of the studied samples. Finally, the expression levels of the target genes in the studied samples as compared with the reference sample were calculated as 2^−ΔΔCt.

Total RNA was extracted with RNAiso Plus (TaKaRa, Dalian, China)), and reverse transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa, Dalian, China). Quantitative real-time PCR analysis was performed with SYBR Premix Ex Taq II reagents from TaKaRa (Dalian, China), according to the manufacturer’s instructions. PCR reactions were performed in a 20 μL mixtures containing 2 μL of cDNA. Aggrecan, type II collagen, MMP-13, COX-2, PGE₂ and β-actin cDNAs were amplified using specific primers (TaKaRa). Gene expression was normalized to glyceraldehyde-3-phosphatedehydrogenase (GADPH) and derived by the 2^-∆∆CT method.

Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Specific primers purchased from TsingKe (Beijing, China) are used as follows (Table S1): cDNA was synthesized using a PrimeScript reagent kit (Takara Bio, Kusatsu, Japan) and was analyzed by qPCR using Real-Time PCR System (ABI 7500, Thermo Fisher Scientific; Waltham, MA, USA) with SYBR Green PCR Master Mix (Thermo Fisher Scientific; Waltham, MA, USA). After an initial denaturation step (95 °C for 4 min), the PCR reaction consisted of 40 cycles (95 °C, 15 s; 57 °C, 15 s; and 72 °C, 1 min). Gene expression level was calculated using the comparative 2^−ΔΔCt method and normalized to that of GAPDH, which was used as an internal control.

To evaluate whether the HIF-1/HRE pathway was activated and the function it served, we measured transcripts of HIF-1α and its downstream genes. At the end of reoxygenation, total RNA from cardiomyocytes was extracted with TRIzol using an RNA extraction kit (TaKaRa, Dalian, China), according to the manufacturer's instructions. The concentration and purity of the total RNA was determined by spectrophotometry at 260 and 280 nm, with an OD₂₆₀/OD₂₈₀ ratio between 1.8 and 2.0 suggesting that the total RNA was pure. Complementary DNA (cDNA) was synthesized from total RNA using the QuantiTect Reverse Transcription kit (TaKaRa). The mRNA levels of HIF-1α, VEGF, Bcl-2, and Bax were measured by RT-PCR, and the following specific primers were used (TaKaRa):