Additional file 1 of Targeting stress induction of GRP78 by cardiac glycoside oleandrin dually suppresses cancer and COVID-19
Creators: name: Ha, Dat P.
name: Shin, Woo-Jin
name: Liu, Ze
name: Doche, Michael E.
name: Lau, Roy
name: Leli, Nektaria Maria
name: Conn, Crystal S.
name: Russo, Mariangela
name: Lorenzato, Annalisa
name: Koumenis, Constantinos
name: Yu, Min
name: Mumenthaler, Shannon M.
name: Lee, Amy S.
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Comparative analysis of GRP78 mRNA expression in human colon and breast tissues. (A) GRP78 mRNA expression in human normal colon (n=349) and colon adenocarcinoma tumor tissues (n=275) from TCGA and GTEx databases. Data analysis was performed by the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) tool. (B) Same as in (A) except the colon adenocarcinoma tumor tissues are divided into subtypes. (C) Same as in (A) except the comparison is made between human normal (n=291) and breast cancer tissues (n=810). (D) Same as in (C) except the breast cancer tissues are divided into subtypes. * p≤ 0.05 (Student's t test). Figure S2. Cardiac glycosides inhibit stress-induction of GRP78 protein in a dose-dependent manner without affecting other chaperones. (A) HCT116 cells were treated with LanC or OLN (from 10 nM to 100 nM) alone or in combination with Tg (300 nM) for 24 hr. WCLs were subjected to Western blot analysis for GRP78 protein level with β-actin serving as loading control. Quantitation of the relative levels of GRP78 normalized to β-actin are shown in the graphs below. (B) Same as in (A) except HT-29 cells were treated with OLN (from 10 nM to 100 nM) alone or in combination with Tg (300 nM) for 24 hr. (C) HCT116 cells were treated with OLN (from 10 nM to 100 nM) alone or in combination with Tg (300 nM) for 24 hr. WCLs were subjected to Western blot analysis for GRP94, HSP70, calnexin, and PDI protein levels with β-actin serving as loading control. (D) Same as in (C) except HT-29 cells were used. Data are presented as mean ± S.D. Figure S3. Lack of suppressive effect on GRP78 stress induction by cardiac glycosides in murine cells and OLN does not affect GRP78 transcript level in α3 knockdown cells treated with Tg. (A) Mouse embryonic fibroblasts (MEFs) or mouse acinar pancreatic cancer cells 266-6 were treated with 1 μM of lanatoside C, digoxin, ouabain, bufalin or 35 nM of oleandrin (OLN) alone or in combination with Tg (300 nM) for 24 hr. Whole cell lysates (WCLs) were subjected to Western blot analysis for GRP78 protein level with β-actin serving as loading control. (B) HCT116 cells were transfected with control siRNA (siCtrl) or siRNA targeting the NKA α3 isoform (siα3) for 24 hr. The cells were then treated with OLN (35 nM) alone or in combination with Tg (300 nM) for an additional 24 hr. Total RNA was extracted and RT-qPCR was performed to measure GRP78 mRNA levels with β-actin mRNA serving as loading control. Data are presented as mean ± S.D. Figure S4. Effect of oleandrin on the UPR pathways. HCT116 cells were non-treated (DMSO) or treated with OLN (35 or 50 nM) alone or in combination with Tg (300 nM) for 24 hr and analyzed for the UPR. For the PERK pathway, the protein levels of the phosphorylated form of eIF2α at Serine 51 (p-eIF2α), ATF4 and CHOP were measured by Western blot with β-actin serving as loading control. For the IRE1 pathway, the splicing of the XBP-1 mRNA transcript was analyzed by PCR with β -actin mRNA serving as loading control. The unspliced transcript is denoted with (u) and the spliced variant is denoted with (s). For the ATF6 pathway, ATF6 protein levels were measured by Western blot. The full-length ATF6 protein is denoted with (FL), and the cleaved version is denoted with (C). Figure S5. Oleandrin reduces GRP78 protein level in MDA-MB-231-GFP/Luc xenograft tumor tissues without majorly affecting other chaperones. (A) Immunohistochemical staining of the xenograft tumor tissues for GRP78 protein level (Scale bar, 100 μm). (B) Western blot analysis of xenograft tumor tissues for GRP94, HSP70, calnexin, and PDI protein levels with β-actin serving as loading control. (C) Quantitation of the relative protein levels normalized to β-actin in (B). Data are presented as mean ± S.D. *p≤ 0.05, n.s. denotes not significant. (Student’s t test). Figure S6. Oleandrin blocks infection by SARS-CoV-2 variants without impacting cell viability and does not affect cell surface ACE2 expression. (A) Confluent monolayers of Vero E6-ACE2 cells were treated with DMSO or increasing concentrations of OLN for 72 hr. Brightfield microscopy images of the cells were taken (Scale bar, 100 μm). (B) Experimental scheme to purify and detect cell surface proteins (Left). Vero E6-ACE2 cells were treated with DMSO or OLN (35 nM) for 48 hr. Whole cell lysate (WCL) and cell surface fractions were subjected to Western blot analysis for GRP78 and ACE2 protein level with GAPDH and Annexin A2 (ANXA2) serving as loading controls for total and cell surface proteins respectively. (C) Confluent monolayers of Vero E6-ACE2 cells in 6-well plates were infected with SARS-CoV-2 Beta or Delta variants and treated with increasing concentrations of OLN as indicated for 72 hr. At the end of treatment, the cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Plaques were counted and plotted as percentage relative to the untreated control in the graph on the left and their size was measured and expressed relative to DMSO-treated control in the graph on the right. (D) A549-ACE2 cells were treated with increasing concentrations of OLN for 48 or 72 hr and cell viability was measure by WST-1 assay. Data are presented as mean ± S.D. Figure S7. Sequence alignment of the putative Spike protein recognition site for GRP78 among different variants of SARS-CoV-2. The amino acid sequences of Spike protein of different variants of SARS-CoV-2 were aligned. The putative Spike recognition site for GRP78 from residues P479-C488 with the highly conserved PCXXXXXXNC sequence is highlighted.