Ac tubulin

Parental and DIAPH3-silenced or over-expressing cell lines, such as DU145, Human Mammary Epithelial (HMEC), or U87, have been described5 (link). Paclitaxel (Cayman Chemicals, Sigma), epothilone B (Cayman Chemicals), and docetaxel (LC Laboratories). Cholera toxin B (CTxB, Sigma, Life Technologies). β-octylglucopyranoside and cacodylate buffer (Sigma), Protein A/G Agarose (Santa Cruz Biotechnology), and Oregon Green-488 Paclitaxel (Invitrogen). Antibodies: Ac-tubulin (Abcam, Cell Signaling), GFP (Genscript), Cleaved PARP, E-cadherin, β-catenin, (Cell Signaling), α-tubulin (Cell Signaling, Genscript), β-tubulin and tubulin βIII (Genscript), DIAPH3 (HNH3.1, a generous gift of Dr. Henry Higgs, Dartmouth Medical School).

HBECs grown in transwell filters as described above were inoculated with 10⁴ PFU of ic-SARS-CoV-2-mNG [59 (link)] or a mock control. HBECs were fixed with 4% PFA for 30 minutes at RT, followed by permeabilization with 0.2% Triton X100 in 1X PBS for 10 minutes at RT. Cells were blocked with 10% normal goat serum in 1X PBS (blocking buffer) for 1 hour at RT. Primary antibodies for Ac-tubulin (Abcam, Cambridge, Massachusetts, USA) and Forkhead Box J1 (FOXJ1) (Sigma Aldrich, St. Louis, Missouri, USA) were diluted in blocking buffer at 1:500 and were incubated overnight at 4°C. Goat anti-mouse Alexa Fluor 594 (BioLegend, San Diego, California, USA) and goat anti-rabbit APC (Invitrogen) were diluted in the blocking buffer at 1:200 and were applied for 2 hours at RT and further stained with Hoechst 33342 (Life Technologies) for 30 minutes at RT. The transwell filters were then cut and placed in a glass slide and mounted with a Prolong Diamond Antifade Mountant (Life Technologies). Representative photos were taken using a Leica LSR microscope. Scale bars correspond to 25 μm.

Protein lysates were isolated from cells or tumor sections using RIPA buffer (CST, Danvers, MA, USA). Protein separation was performed using electrophoresis in 10–15% arc-bis gels, and the proteins were transferred onto PVDF using a transfer system (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the appropriate primary antibodies (1:1000) and secondary antibodies (Thermo, A11034, A32723 1:10000), reacted with ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA), and incubated for several minutes in a dark room. Antibodies against PD-L1 (Cell Signaling Technology, 60475, 1:1000), NAMPT (Proteintech, MA5-24108, 1:1000), β-actin (Abcam, ab8226, 1:1000), GAPDH (Abcam, ab8245, 1:1000), Ac-tubulin (Abcam, ab24610, 1:1000), α-Tubulin (Abcam, ab7291, 1:1000), Ac-NF-κB p65 (K310) (Thermo, PA5-17264, 1:1000), NF-κB p65 (Thermo, PA5-23170, 1:1000), HIF-1α (Thermo, MA1-516, 1:1000) were purchased from commercial company.