Renilla luciferase control reporter vector

Cells were transfected with a firefly luciferase reporter vector and Renilla luciferase control reporter vector (Promega) using Lipofectamine LTX, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After 24 h, luciferase activity was measured using a dual‐luciferase reporter assay system (Promega, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity. To investigate the effects of CSE, nicotine, BaP, and inflammatory factors on the activity of transcriptional elements, cells were treated with the compounds or inflammatory factors for 12 h prior to the luciferase assay. Cells in the control group were incubated with 0.1% DMSO (Sigma, Santa Clara, CA, USA). To investigate the effects of transcription factors on the activity of transcriptional elements, cells were transfected with small interfering RNA (siRNA) targeting NR4A2 for 24 h prior to stimulation and luciferase assays. siRNAs targeting NR4A2 and the scrambled sequence (siNR4A2, siScramble) were synthesized by RiboBio (Guangzhou, China). All luciferase assays were repeated thrice with at least three replicates. The siRNA sequences are listed in the Supporting Information.

We transiently transfected HEK293 cells with the SBE-luciferase vector (pGL4.48 [luc2P/SBE/Hygro], Promega, Madison, WI, USA) along with the Renilla luciferase control reporter vector (pRL, Promega), which allowed us to normalize firefly luciferase activities according to transfection efficiency. The following day, we treated the transfected HEK293 cells with rh-TGF-β1 (1 nM) or vehicle (PBS) in the presence and absence of step-tag-fortilin (3 nM) in quadruplicate, incubated the samples for 18 h at 37 °C, and subjected the cells to the Dual-Glo-Luciferase Assay System (Promega) according to the manufacturer’s instructions. We calculated relative luciferase activity (FLU/RLU) by dividing firefly luciferase units (FLU) by Renilla luciferase units (RLU) and expressed the results as A.U. We then normalized FLU/RLU of the cells treated with rh-TGF-β1 to that of the cells treated with vehicle in either the presence or absence of fortilin.

A total of 20,000 HEK293T cells were plated into each well of a poly-l-lysine–coated 96-well flat-bottom plate and allowed to adhere overnight. A constitutive cytokine receptor vector (2.5 ng), a STAT response element vector that drives Firefly luciferase (100 ng; Promega, Madison, WI) and Renilla luciferase control reporter vector (1 ng; Promega, Madison, WI) were mixed in a final volume of 5 μl in Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA) (“DNA mix”). Lipofectamine 2000 (0.3 μl; Invitrogen, Thermo Fisher Scientific, Waltham, MA) in 5 μl of Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mix. The mixture was incubated at room temperature for 20 min, and the total volume of 10 μl was added to each well containing HEK293T cells. Twenty-four hours after transfection, cells were left untreated, treated with AP1903 (1 μg/ml; APExBio, Houston, TX), or with the indicated cytokines diluted in serum-free media. At the indicated time points after treatment, reporter activity was determined using the Dual-Glo Luciferase Assay System (Promega, Madison, WI) as per the manufacturer’s instructions. Firefly luciferase activity was first normalized to Renilla luciferase activity, and then STATresponse element fold induction was calculated by normalizing to untransfected controls.