Fig 1: Combination treatment with RCM-1 and venetoclax reveals a unique genetic profile in 76-9 cells. (A) Venn diagrams illustrated the overlap of differentially expressed genes between the combination therapy and venetoclax compared with the control. (B) Gene set enrichment analysis of RNA-sequencing data highlighted the upregulated pathways in the combination therapy relative to venetoclax, such as ferroptosis and elevated cytosolic Ca+2 level, as well as the downregulated pathways, such as WNT signaling pathway, mitotic cell cycle and angiogenesis pathway. (C) Volcano plot identified the differentially expressed genes in the combination therapy vs. venetoclax. Atp2b4 was notably downregulated in the combination therapy. (D) Reverse transcription-quantitative PCR showed the downregulation of Atp2b4 in 76-9 cells treated with RCM-1 and combination therapy. Actb mRNA was used for normalization (E) Protein levels of ATP2B4 were decreased after RCM-1 and the combination treatments, as shown by immunofluorescence using ATP2B4 antibodies. A total of five random fields per sample were used to quantify the percentage of ATP2B4-positive cells per group. Scale bar, 10 μm. Data presented as mean ± SD. **P≤0.01, ***P≤0.001, ****P≤0.0001.
Fig 2: The knockdown of ATP2B4 slows RMS tumor growth. (A) Control and shAtp2b4 76-9 cells were inoculated subcutaneously into the flank of C57Bl/6J mice. Tumor volume measured at different time points. Knockdown of ATP2B4 significantly impaired tumor growth compared with control mice (n=5 per group; presented as the mean ± SD). (B) Tumor volumes measured after tumor harvest show smaller shAtp2b4 tumors. The average vehicle tumor volume was 1,321 mm3 compared with 592 mm3 for shAtp2b4 C and 468 mm3 for shAtp2b4 D. The maximum tumor diameter was 15.56×13.39 mm and the corresponding maximum tumor volume was 1,395 mm3. (C) Gross images of control and shAtp2b4 RMS tumors. (D) Reverse transcription-quantitative PCR analysis of mRNA isolated from tumors confirmed ~50% knockdown efficiency in shAtp2b4-expressing tumors. *P≤0.05, **P≤0.01, ****P≤0.0001. ATP2B4; ATPase Plasma Membrane Ca2+ Transporting 4; RMS, rhabdomyosarcoma; sh, short hairpin.
Fig 3: Overexpression of ATP2B4 increases the resistance of RMS cells to apoptosis. (A) Representative immunofluorescence images depicting control, pATP2B4a and pATP2B4b 76-9 cells treated with DMSO, 5 mM, or 8 mM of venetoclax for 24 h. The overexpression of both ATP2B4 isoforms reduced apoptosis compared with control RMS cells following venetoclax treatment. Scale bar, 100 mm. (B) The percentage of positive annexin and DEVD cells were counted in five random fields using the EVOS imaging software and presented as the mean ± SD from triplicates of one experiment. ***P≤0.001, ****P≤0.0001. ATP2B4; ATPase Plasma Membrane Ca2+ Transporting 4; RMS, rhabdomyosarcoma.
Fig 4: ATP2B4 knockdown enhances venetoclax-mediated apoptosis in 76-9 RMS cells. (A) Representative immunofluorescence images from control and siATP2B4 76-9 cells treated with DMSO, 5 and or 8 mM of venetoclax for 24 h. Venetoclax-induced dose-dependent apoptosis was measured by caspase 3/7 (DEVD) activity (red) and annexin V (green). Scale bar, 100 mm. The knockdown of ATP2B4 increased RMS apoptosis compared with control cells. (B) The percentage of annexin and DEVD positive cells were counted in five random fields using the EVOS imaging software and presented as mean ± SD from triplicates of one experiment. **P≤0.01, ***P≤0.001, ****P≤0.0001. ATP2B4; ATPase Plasma Membrane Ca2+ Transporting 4; RMS, rhabdomyosarcoma.
Fig 5: ATP2B4 is differentially overexpressed in RMS cells vs. normal muscle cells and its knockdown decreases tumor cell proliferation, migration and colony formation. (A) Left panel, Human myoblast (muscle progenitor cells), RMS, normal skeletal muscle and smooth muscle scRNA sequencing datasets were visualized using UMAP. Data were extracted from GSE143704 for normal muscle tissue, GSE 195709 for RMS (RMS1 and 3 are fusion-negative RMS, RMS2 is fusion-positive and RMS4 is spindle (fusion-negative). Right panel, ATP2B4 mRNA expression was higher in RMS cells compared with normal muscle cells. (B) Reverse transcription-quantitative PCR showed the upregulation of ATP2B4 mRNA level in RMS cells (RD and RH30) compared with HSkMC. (C) Knockdown of ATP2B4 increased the intracellular calcium level. Calcium level was assessed using the calcium-sensitive fluorescent dye fura-2 AM. Scale bar, 10 μm. (D) Knockdown of ATP2B4 inhibited colony formation in 76-9 RMS cells. Data presented as mean ± SD. (E) Knockdown of ATP2B4 suppressed 76-9 cell proliferation in culture. Data presented as mean ± SD. (F) ATP2B4 knockdown decreased RMS cell migration. The percentage of wound closure presented as mean ± SD (magnification, ×4). *P≤0.05, **P≤0.01, ***P≤0.001. RMS, rhabdomyosarcoma; UMAP, Uniform Manifold Approximation and Projection; HSkMC, human skeletal muscle cells; SMC, smooth muscle cells.
Supplier Page from OriGene Technologies for Atp2b4 Mouse shRNA Lentiviral Particle (Locus ID 381290)