Cell culture and treatment

The CFBE-WT and the CFBE-dF cells were gifts from Dr. Fei Sun’s laboratory at Wayne State University [65]. The CFBE-WT cells were cultured in MEM media (#11095-80, Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (#10,438,026, Gibco, Waltham, MA, USA) and 0.5 µg/ml puromycin (#P9620, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO2 in a humidified incubator. The CFBE-dF cells were cultured in MEM media with 10% FBS and 1 µg/ml puromycin. The CFPAC-1 (#CRL-1918) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in IMDM media (#12,440,053, Gibco, Waltham, MA, USA) with 10% FBS and 100U/ml penicillin/streptomycin (#15,140,122, Gibco, Waltham, MA, USA).

Human kidney 2 (HK-2) cells were obtained from ATCC (#CRL-2190), and were cultured in DMEM-F12 media (#11,320,033, Gibco, Waltham, MA, USA) with 10% FBS.

The hepatocarcinoma cell line Huh-7 was originally provided by Dr. Charles Rice at Rockefeller University [66] and cultured in DMEM/High Glucose media containing 10% FBS.

To evaluate the effects of Sotagliflozin and Rapamycin, the CFBE-WT cells and the CFBE-dF cells were treated with 20µM Sotagliflozin (#17,011,711, Sun-Shine Chemical Technology Co., Ltd, Shanghai, China.) in serum-free MEM media for 24 h, or were treated with 20nM Rapamycin (#S1039, SelleckChem, Houston, TX, USA) in serum-free MEM media for 48 h.

To assess the effects of Sotagliflozin on attenuating hepatic ER stress response and elevation of SGLT1, Huh-7 cells were treated with 10 µg/mL fatty acid-free BSA-complexed palmitate acids (PA, #P0500, Sigma-Aldrich, St. Louis, USA) in the presence or absence of Sotagliflozin (50 µg/mL) for 36 h.

Airway epithelial cells from CF patients and healthy control individuals

Human airway basal cells from healthy control (HC) and CF patients carrying the CFTR-F508del homozygous mutation were isolated from freshly discarded lung tissues at Massachusetts General Hospital under Institutional Review Board (IRB) protocol approval (#2017P001479 and #2013P002332). The airway basal cells were used to generate matured airway epithelial cells in the air-liquid interface (ALI) culture, as previously described [67]. The matured airway epithelial cells from HC and CF individuals were used for the analysis of gene and protein expressions. to promote squamous remodeling, 1 µM CHIR99021 (#4423, Tocris Bioscience, Minneapolis, MN) was added to the ALI medium as previously described [47]. After 14 days of differentiation, epithelial cells were treated with vehicle, 20 µM Sotagliflozin and 10 nM Rapamycin, individually or in combination, for 3 days before cell harvest for protein and RNA extraction.

Antibodies

Antibodies against Bip/GRP78 (#3177), IRE1α (#3294), and β-actin (#3700) were obtained from Cell Signaling Technologies (Danvers, MA, USA). The CFTR antibody (#217 and #596) was from the Cystic Fibrosis Foundation Therapeutics (Bethesda, MD). The SGLT1 antibodies were from Invitrogen (PA5-88282, Waltham, MA, USA; for Western blot) and Abcam (ab14686, Waltham, MA, US; for immunofluorescence staining). The p-IRE1α antibody (#AP0878) was from ABclonal Technology (Woburn, MA, USA). The XBP1s antibody (#619,502) was from BioLegend (San Diego, CA, USA). The secondary antibodies were from LI-COR Biosciences (#D01216-10 and #D00226-05, Lincoln, NE, USA; for Western blot) and Jackson ImmunoResearch Laboratories (#147,158, West Grove, PA, USA; for immunofluorescence staining).

Protein extraction and Western blot

Cells were lysed in RIPA lysis buffer (#89,900, ThermoFisher Scientific, Waltham, MA, USA) supplemented with protease inhibitor and phosphatase inhibitor cocktails (#11,873,580,001, Roche, Penzberg, Germany). Proteins were resolved in 10% SDS–PAGE gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in TBST containing 5% non-fat milk at room temperature for 2 h, and were incubated with primary antibodies (1:1000 dilution) at 4 °C overnight. After washing with TBST, membranes were incubated with secondary antibodies (1:8000 dilution) at room temperature for 1 h. After TBST wash, bands were scanned and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA from cell samples was extracted using the Trizol reagent (#15,596,018, ThermoFisher Scientific) and purified with the RNeasy kit (#74,106, QIAGEN, Hilden, Germany). RNAs were reverse transcribed into cDNA with the SuperScript III kit (#18,080,051, ThermoFisher Scientific). The target gene expression at the transcription level was assessed by the quantitative real-time PCR system (Bio-Rad, Hercules, CA, USA) using iQ SYBR Green Supermix (#1,708,884, Bio-Rad, Hercules, CA, USA). β-actin was used as the internal control. Primers for qRT-PCR are listed in Supplemental Table 2.

Immunofluorescence (IF) staining

The CFBE-WT cells and the CFBE-dF cells were fixed on Nunc Lab-Tek Chamber Slides (Thermo Fisher Scientific) with 4% paraformaldehyde in PBS for 15 min. After washing with PBS, the slides were blocked with 5% donkey serum for 1 h at room temperature and then incubated with primary antibody against SGLT1 (1:50 dilution). After PBS washing, the slides were incubated with Alexa Fluor–labeled secondary antibody (1:1000 dilution) at room temperature for 1 h. IF slides were washed with PBS and mounted before image collection. IF images were acquired using an Olympus IX73 microscope.

Adenoviral infection

Adenovirus expressing spliced XBP1 (Ad-XBP1s) and adenovirus expressing mutant IRE1 K907A (Ad-K907A) were produced as previously described [32]. For XBP1s overexpression, the CFBE-WT cells and the CFBE-dF cells at approximately 70–80% confluency were infected with Ad-XBP1s at a MOI of 100 for 48 h. To suppress XBP1 expression, the cells were transduced with Ad-K907A at an MOI of 100 for 48 h. The adenovirus encoding LacZ (Ad-LacZ) infection or non-infection served as controls.

Chromatin immunoprecipitation (ChIP) assay

The CFBE-WT cells and the CFBE-dF cells were transduced with Ad-XBP1s at an MOI of 100 for 48 h to overexpress XBP1s. Ad-LacZ was used as the vehicle control. ChIP assay was performed with the SimpleChIP® Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technologies, Danvers, MA, USA), following the manufacturer’s instructions. The DNA/protein complex was immunoprecipitated with control IgG or with anti-XBP1s antibody. Purified precipitated DNA was used as the template for qPCR. Primers used to detect the putative XBP1s binding motif on human SLC5A1 promoter are listed in Supplemental Table 2.

Plasmid construction and transfection

The putative promoter region of the human SLC5A1 gene (from position − 1087 to position − 21) was PCR-amplified from human genomic DNA by using forward primer 5’-TTTCTGTGGTCCTCTGCCTC-3’ and reverse primer 5’-TCCTTATACGGCCTCCTGGT-3’. The amplified promoter region was inserted into the pGL4.10 luciferase reporter vector (#E1910, Promega, Madison, WI, USA) by using In-Fusion® HD Cloning Kit (#102,518, Takara, CA, USA). The 5’-CCACCCACCCACC-3’ box on the human SLC5A1 promoter (-595 to -583), which is the putative XBP1s binding site, was mutated to 5’-TACAGACTAATGA-3’ by using Q5 SiteDirected Mutagenesis Kit (#E0554S, New England Biolabs, Ipswich, MA). All plasmids were validated by Sanger sequencing. The plasmids were named wt-Luc and mut-Luc, respectively.

The CFBE-WT cells and the CFBE-dF cells at 70–80% confluence were transfected with either the wt-Luc or the mut-Luc plasmids by lipofectamine 2000 (#11,668,019, ThermoFisher Scientific) according to the manufacturer’s protocol. After 24 h, these cells were infected with Ad-LacZ or Ad-XBP1s (MOI, 100). Luciferase activity was detected using Dual Luciferase Reporter Assay System (#E1910, Promega, Madison, WI, USA).

Statistics analysis

Statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Data were reported as mean ± SEM (standard error of means) with three replicates for each data point. Comparison between two groups was analyzed by unpaired, 2-tailed Student’s t test (GraphPad).