Mm00517691 m1

Total RNA was extracted by the RNAqueous^®-4PCR kit (Ambion, Austin, TX, USA), then was used to synthesize cDNA. The cDNA was analyzed by the™ QuantStudio^® 3D Digital PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was first loaded onto the chips using the QuantStudio^® 3D Digital PCR Chip Loader with a mixture comprising 2× Quantstudio^® 3D digital PCR mastermix, and also 300 nmol/L of GRP78 primers and probes (Mm00517691_m1; Applied Biosystems, Foster City, CA, USA). Next, the chips were sealed and loaded onto a GeneAMPR PCR system 9700 (Applied Biosystems, Foster City, CA, USA), and then the following cycling conditions were used: 96 °C for 10 min, followed by 39 cycles of 60 °C for 2 min and 98 °C for 30 s, and a final extension at 60 °C for 2 min. Finally, the end-point fluorescence of the segmentation processes on the chips was analyzed by transferring the chips to the measurement unit (application version 1.1.3, algorithm version 0.13, Applied Biosystems, Foster City, CA, USA). In addition, the mRNA signal of GRP78 was normalized to18S rRNA.

RNeasy Mini Kit (QIAGEN) was used to prepare total RNA from mouse tissues and cultured cells, and ReliaPrep RNA Tissue Miniprep System (Promega) from human samples. Reverse-transcription reaction was carried out with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), after treatment with DNAse (Promega). Quantitative PCR analyses were performed using ABI Prism 7900, with TaqMan Gene Expression Master Mix or Power SYBR Green PCR Master Mix (Applied Biosystems). The relative expression levels were normalized by the expression level of Ppia (encoding Cyclophilin A) in samples of mouse tissues and cultured cells, control IgG and control upstream region in ChIP assays, and GAPDH in human samples, respectively. TaqMan Assays were purchased from Applied Biosystems: Hspa5 (BiP), Mm00517691_m1; Ddit3 (CHOP), Mm00492097_m1; Sdf2l1, Mm00452079_m1; Ero1lb, Mm00470754_m1; Pdia3, Mm00433130_m1; Sdf2, Mm00485985_m1; Pparg, Mm00440945_m1; Pten, Mm00477208_m1; Sod1, Mm01344233_g1; Sod2, Mm01313000_m1; Tnf, Mm99999068_m1; Ppara: Mm00627559_m1; Fasn, Mm00662319_m1; Acacb, Mm01204671_m1; Pck1: Mm00440636_m1.