Realsybr mixture

Total RNA was extracted according to the miRNeasy Micro Kit (Qiagen) manufacturer's protocol from cells harvested by LCM. Total RNA was dissolved in 14 µL of diethyl pyrocarbonate-treated H₂O. All RNA was verified for purity by measuring the ratios of the absorbance at 260 nm and 280 nm (A260/A280) using a spectrophotometer. All the RNA was reverse-transcribed in a final volume of 20 µL using a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa Bio Inc, Shiga, Japan) according to the protocol. Quantitative analysis of GILT mRNA expression was performed in paired breast cancer cells and normal epithelial cells using the RealSYBR Mixture (CWBIO, Beijing, People's Republic of China). GILT was amplified using the following primers: 5′-TGACCCTCTACTATGAAGCACTG-3′ (forward primer) and 5′- CCACTGACATTTTGTTCCTGTG-3′ (reverse primer). ACTB was used as an endogenous control with the following primers: 5′-TGACGTGGACATCCGCAAAG -3′ (forward primer) and 5′-CTGGAAGGTGGACAGCGAGG-3′ (reverse primer). The results were evaluated by the comparative threshold cycle value method (2^−Δct) for relative quantification. Each reverse transcriptase (RT)-qPCR experiment was repeated in triplicate.

Liver cancer cells were lysed using RIPA buffer (Cell Signal Technology, MA), centrifuged for the supernatant. The protein concentration was measured using bicinchoninic acid (BCA) assay (Cwbio, Beijing, China). The lysates were then diluted in loading buffer and denatured by heating at 100°C. Standard Western blot assay were performed using DLAT antibody (Proteintech, 68303) and GAPDH antibody (Abcam, ab77109) as the loading control.
Total RNA was extracted from the liver cancer cells using Trizol reagent (Invitrogen, USA) and cDNA was synthesized using the M-MLV Reverse Transcriptase Kit (Cwbio) following the manufacturer’s instructions. RT-PCR was performed using Real SYBR Mixture (Cwbio) on a Lightcycler 480 II instrument (Roche Applied Science, USA). GAPDH severed as the internal control.

Liver cancer cells were lysed using RIPA buffer (Cell Signal Technology, MA), centrifuged for the supernatant. The protein concentration was measured using bicinchoninic acid (BCA) assay (Cwbio, Beijing, China). The lysates were then diluted in loading buffer and denatured by heating at 100 ℃. Standard Western blot assay were performed using NUSAP1 antibody (Proteintech, 12024-1-AP) and GAPDH antibody (Abcam, ab77109) as the loading control.
Total RNA was extracted from the liver cancer cells using Trizol reagent (Invitrogen, USA) and cDNA was synthesized using the M-MLV Reverse Transcriptase Kit (Cwbio) following the manufacturer’s instructions. RT-PCR was performed using Real SYBR Mixture (Cwbio) on a Lightcycler 480 II instrument (Roche Applied Science, USA). GAPDH severed as the internal control.
NUSAP1 forward primer: 5ʹ-AGCCCATCAATAAGGGAGGG-3ʹ;
NUSAP1 reverse primer: 5ʹ-ACCTGACACCCGTTTTAGCTG-3ʹ.
GAPDH forward primer: 5ʹ-TGTTGCCATCAATGACCCCTT-3ʹ;
GAPDH reverse primer: 5ʹ-CTCCACGACGTACTCAGCG-3ʹ.

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with the M‐MLV Reverse Transcriptase Kit (Cwbio, Beijing, China). PCR analyses were conducted to quantify mRNA expression using Real SYBR Mixture (Cwbio) on a Lightcycler 480 II instrument (Roche Applied Science). GAPDH severed as an internal control. The specific oligonucleotide primer pairs are listed in Table S3, Supporting Information.