Blood cell total rna mini kit

For gene expression analysis, 40–50 mg liver sample was homogenized with a micropestle and syringe in Qiazol Lysis Reagent (QIAGEN, Germany). Total RNA was purified using a Blood/Cell Total RNA Mini Kit (Geneaid, Taiwan). The quantity of the RNA was measured using a Nanodrop-1000 (ThermoFisher, USA). We used SensiFAST cDNA Synthesis Kit (Bioline, UK) to prepare cDNA from 1 µg of total RNA. Quantitative real-time PCR (RT-qPCR) was performed on the 7900 HT Fast Real-Time PCR System (Applied Biosystems, USA), using SensiFAST Probe Hi-ROX Kit (Bioline, UK) according to the instructions of the manufacturer. Briefly, each reaction was performed in the final volume of 10 µL, including 50 ng of cDNA (in 4.5 µL), 0.5 µL of Taqman assay and 5 µL of SensiFAST Probe Hi-ROX mix. Taqman assays were used for Gapdh (Mm9999915_g1), Cd36 (Mm00432403_m1), Ldlr (Mm01177349_m1), Anxa2 (Mm00500307_m1) and Pcsk9 (Mm01263609_g1). The fold changes of mRNA were calculated using 2^−ΔΔCt method, normalized to Gapdh RNA internal control.

Blood/Cell Total RNA mini kit from Geneaid used to isolate RNA from 10⁶ cells. Subsequently, the RNA synthesized to cDNA. The primer sequence for Smad7 is mentioned as follows: Smad7 Fwd: 5′- AAACAGGGGGAACGAATTATC-3′; Smad7 Rev: 5′- ACCACGCACCAGTGTGAC-3′. On the other hand, the primer sequence for other gene were quoted from other previous studies: β-actin and Smad3 from Rahmaniah et al,^{13 (link)} TGF-β1 and TGF-β1-Receptor from Lestari et al,^{14 (link)} and E-cadherin and vimentin from Paramita et al.^{20 (link)} The qRT-PCR was done using Toyobo Thunderbird SYBR qPCR Mix (Japan) according to manufacturer protocol. Reactions were performed in qRT-PCR Light Cycler Nano Roche^TM. Temperatures in this study are set to incubation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 20 seconds and annealing temperature for 58°C–60°C for 1 minute. All quantification cycle (Cq) will be processed according to Livak method.^{20 (link)}

Human HCC cells, HepG2, were gifted from the Eijkman Institute for Molecular Biology Laboratory, Indonesia. Sorafenib and alpha mangostin were purchased from St. Cruz Biotechnology. Dimethylsulfoxide was from Sigma Aldrich (Singapore). TGF-β1 ELISA kit was from R&D System (USA). Dulbecco’s Minimal Essential Medium (DMEM), fetal bovine serum - heat-inactivated, Fungizone, and Penicillin/Streptomycin were obtained from Gibco (USA). MTS assay kit was obtained from Promega (USA). Blood/Cell Total RNA mini kit was purchased from Geneaid (USA). ReverTra Ace quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) master mix with gDNA remover and Thunderbird SYBR qPCR Mix was from Toyobo (Japan). Cell lysis buffer was obtained from Invitrogen (USA). Primers used were purchased from First Base (Singapore). Primary antibodies E-cadherin, β-actin, and secondary antibody anti-rabbit IgG HRP-linked antibody were from Cell Signaling Technology (USA).