β actin

Total RNA from NSCLC cells was isolated using RNAiso Plus extraction reagent and cDNA was produced using PrimeScript Reverse Transcriptase (Takara Bio, Inc., Otsu, Japan) according to the manufacturer’s instructions. RT-qPCR reactions were then conducted with SYBR Premix Ex Taq II (Takara Bio, Inc.) in 25 μl reactions with 2 μl cDNA and 0.4 μM each of the forward and reverse primers using the following thermocycling conditions: 95°C for 30 sec, 95°C for 5 sec and 60°C for 30 sec. Data was collected over 40 cycles and all expression levels were normalized to β-actin. The following primers were used: Gli1, F 5′-CTG GAC CTG CAG ACG GTT ATC-3′ and R 5′-AGC CTC CTG GAG ATG TGC AT-3′; β-actin, F 5′-TGA CGT GGA CAT CCG CAA AG-3′ and R 5′-CTG GAA GGT GGA CAG CGA GG-3′ (Sangon Biotech Co., Ltd., Shanghai, China).

Total RNA was extracted using the RNeasy Plus Mini Kit (50) (Qiagen, Hilden, Germany), and miRNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Then, cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa, Dalian, China). The SYBR Premix Ex Taq II (TaKaRa) was used to amplify the double-stranded cDNA of interest. qPCR primers for miR-520b and U6 were purchased from RuiBo Bio (Guangzhou, China). qPCR primers for GATA6, CREB1 and ACTB (β-actin) were synthesized by TaKaRa (Dalian, China). The levels of U6 and ACTB were used as internal controls for miRNA and mRNA, respectively. The 2^–ΔΔCt method was used to determine the relative expression level of RNA between groups. The primer sequences are listed in Supplementary Table S1.

The cells were digested with trypsin, washed three times with PBS solution, added with an appropriate amount of protein extract (Beijing Soleibo Technology Co., Ltd., China), and shaken in an ice bath for 15 min. After centrifugation (10,000 × g, 4°C) for 20 min, the supernatant was collected and aliquoted into EP tubes, followed by protein purity determination via the BCA kit (Abcam, USA) method. Afterward, 30 μg of protein was subjected to SDS–PAGE (Thermo Fisher, USA) and transferred onto a PVDF membrane (Sigma–Aldrich, USA), followed by 2 h of immersion in 5% skim milk and subsequent overnight incubation with antibodies against Bax (1 : 1000), Caspase-3 (1 : 1000), and β-actin (1 : 1000) (TaKaRa, Japan) at room temperature. The next day, the membranes were washed three times with TBST, followed by a 1 h incubation with the secondary antibody (1 : 2000). Subsequently, the membranes were also washed three times with TBST, followed by exposure and development. A gel imaging system (iBright, Thermo Fisher, USA) and the ImageJ software (National Institutes of Health) were used to analyze the gray value and calculate the relative protein expression.