Sparc

For immunostaining, the following antibodies were used: Oct-4 (Santa Cruz; sc-5279), proSP-C (Sigma-Aldrich; AB3786), AQP5 (Sigma-Aldrich; AB15858), E-cadherin (BD Bioscience; 610182), collagen type I (Sigma-Aldrich; AB765P), phycoerythrin (PE)-conjugated anti-CD157 (BioLegend, 140203), FITC-conjugated anti-CD54 (BD Bioscience, 561898), and allophycocyanin (APC)-conjugated anti-CD45 (BioLegend, 103111). For Western blot and immunoprecipitations, the following antibodies were used: SPARC (Cell Signaling Technology; #5420, #8725), collagen type I (Sigma-Aldrich; AB765P; Santa Cruz, sc-59772), and a β-actin (Sigma-Aldrich; A5441). Secondary antibodies conjugated to HRP were purchased from Jackson ImmunoResearch. Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647 were purchased from Life Technologies.

Western blot was performed as previously described (Li et al., 2020 (link)). The primary antibodies used in this study included CPLA_2α (1:1000, SC-438, Santa Cruz Biotechnology, Dallas, TX), phosphorylated CPLA_2α (1:1000), TNC (1:1000), SPARC (1:1000), α-SMA (1:1000, 19,245T, Cell Signaling Technology), COX-2 (1:1000, 12,282T, Cell Signaling Technology), PGIS (1:1000, 100023, Cayman Chemical), PPARδ (1:1000, ab178866, Abcam), BMP2 (1:1000, A0231, Abclonal, Wuhan, China), WNT4 (1:1000, sc-376279, Santa Cruz Biotechnology), E2F8 (1:1000, A1135, Abclonal), CYCLIN D3 (1:1000, 2936T, Cell Signaling Technology), TUBULIN (1:1000, 2144 S, Cell Signaling Technology), GAPDH (1:1000, sc-32233, Santa Cruz Biotechnology). After the membranes were incubated with an HRP-conjugated secondary antibody (1:5000, Invitrogen) for 1 hr, the signals were detected with an ECL Chemiluminescent Kit (Millipore, USA). Each experiment was repeated at least three times.

To verify the infection status of cells, Western blot analyses were performed to probe for ZIKV non-structural protein NS1 (BioFront Cat. # BF-1225). Proteins from ZIKV-infected and mock-infected samples were separated by SDS-PAGE and transferred to 0.2 μm nitrocellulose membranes. Membranes were blocked in 5% skim milk for 1 h and incubated for 90 min with an antiserum against viral NS1 protein. Detection of ß-actin (Cell Signaling, Cat. # MABT144) served to estimate equal amounts of protein loaded and to normalize expression intensities of proteins. Selected dysregulated host proteins also were examined to verify mass spectrometry (MS) data. The proteins detected included SPARC (Cell Signaling, Cat. # 5420), BAD (Cell Signaling, Cat. # 9292), NF-κB2 (Cell Signaling, Cat. # 4882), DDX5 (Cell Signaling, Cat. # 9877), and EIF4A1 (Cell Signaling, Cat. # 9292). Primary antibody reactivity was detected with HRP-conjugated secondary antibodies (Cell Signaling).

Fibroblasts were collected by trypsinization, washed with 3% bovine serum albumin (BSA) in PBS, and then incubated with Ghost Dye Red 780 Viability Dye stain (1:1000, Tonbo Bioscience, 13-0865-T100) for 10 min at room temperature. Cells were washed with 3% BSA in PBS, fixed with 0.1% paraformaldehyde for 15 min at room temperature, washed again with 3% BSA in PBS, and permeabilized with 2% saponin for 15 min at room temperature. Cells were then incubated with Fc blocking anti-mouse CD16/CD32 antibody (1:100, clone 2.4G2; Tonbo Bioscience, 70-0161-M001) for 10 min. After an additional 3% BSA in PBS wash, cells were labeled with primary antibodies to fibroblast-specific protein 1 (FSP1, Millipore, 07-2274), fibroblast activation protein α (FAP, R&D Systems, MAB9727), α smooth muscle actin (αSMA, Millipore, A2547), platelet-derived growth factor receptor α (PDFGRα, Cell Signaling, 3174), platelet-derived growth factor receptor β (PDGFRβ, Cell Signaling, 3169), Vimentin (Cell Signaling, 5741), or secreted protein acidic and rich in cysteine (SPARC, Cell Signaling, 8725) for 30 min at 4 °C. Again, cells were washed with 3% BSA in PBS and then followed by incubation with appropriate fluorescent secondary antibodies for 30 min at 4 °C. Cells were washed with 3% BSA in PBS three times prior to analysis on a flow cytometer (BD LSRFortessa).

The human pancreatic cancer cell lines PANC-1 and CFPAC-1 were cultured in Dulbecco's modified Eagle's medium and the cholangiocarcinoma cell lines HuCCT-1 and SCK were cultured in RPMI-1640. PANC-1 and CFPAC-1 cells were purchased from the ATCC (Manassas, VA, USA). HuCCT-1 and SCK cells were procured from the Health Science Research Resources Bank (Osaka, Japan) and Dr. Dae-Ghon Kim of Chonbuk National University Medical School and Hospital (Jeonju, Korea), respectively. All cell lines were maintained in a humidified incubator at 37°C with 5% CO₂. Antibodies against S6K, phospho-S6K, S6, phospho-S6, 4EBP1, phospho-4EBP1, cleaved caspase-3, CHOP, Bax, Bim, BCl-2, cyclin B1, HIF-1β, CD44, SPARC, vimentin, and GAPDH were obtained from Cell Signaling Technology. CD-31 and VEGF were purchased from Abcam (Cambridge, MA, USA). HIF-1α, VEFGR2/Flk-1, and MMP-2 were obtained from Santa Cruz Biotechnology.