Journal: Oncology Reports

Article Title: Depleting HIF-1α attenuates the progression of osteosarcoma, but tumorigenicity is sustained through HIF-independent pathways

doi: 10.3892/or.2026.9099

Figure Lengend Snippet: Different survival pathways on which osteosarcoma cells rely in the absence of HIF-1α. Viability of Mock and HIF-KO cells treated for 2 days with 1 µM of (A) the indicated reagents or (B) metformin. Assays were performed in quadruplicate, and data are expressed as the mean ± SD relative (−fold) to the corresponding control value for cells incubated in the absence of these reagents. (C) Viability of Mock and HIF-KO cells treated for 2 days with 1 µM of the indicated reagents. Assays were performed in quadruplicate. (D) Viability of Mock and HIF-KO cells treated for 2 days with simvastatin. Cells were also co-treated with mevalonate (200 µM) and simvastatin. Assays were performed in quadruplicate. (E) Viability of Mock and HIF-KO cells treated for 2 days with 0.5 µM simvastatin or 2 mM metformin. Cells were also co-treated with metformin plus simvastatin. Assays were performed in quadruplicate. (F) Western blot analysis to detect expression of the indicated molecules in Mock and HIF-KO cells cultured for 17 h in the presence/absence of 0.5 µM simvastatin, 200 µM mevalonate and 2 mM metformin under adherent normoxic conditions. Arrows indicate the cleaved active form of Caspase 3. The relative (fold) values of p-p38 or cleaved-Caspase3 against the corresponding control value normalized to the intensities of p38 or Caspase3 bands are shown. (G) Reverse transcription-quantitative PCR analysis of Hmgcr mRNA levels in Mock and HIF-KO cells cultured for 20 h under adherent normoxic or hypoxic conditions (n=3). (H) Viability of Mock and HIF-KO cells treated for 2 days with 1 µM simvastatin alone or co-treated with 200 µM mevalonate, 50 µM FPP or 50 µM GGPP under adherent normoxic conditions. Assays were performed in quadruplicate. (I) Western blot analysis of GTP-RhoA and RhoA in Mock and HIF-KO cells treated with 1 µM simvastatin for 4.5 h. GTP-RhoA was isolated with beads conjugated with a GST fusion protein containing the Rho binding domain of Rhotekin that specifically binds to GTP-RhoA. A part of the same lysate was used for evaluation of total RhoA. The relative (fold) values of GTP-RhoA against the corresponding control value normalized to the intensities of α-Tubulin bands are shown. (J) Proposed model for the heterogeneity of pathways relied on by osteosarcoma cells after HIF-1α depletion. HIF-KO, hypoxia inducible factor knockout. *P<0.05 and **P<0.01.

Article Snippet: Roxadustat (25 μM; 27 h), buparlisib (1 μM; 2 days) (MedChemExpress), antimycin A (1 μM; 2 days), oligomycin (1 μM; 2 days) (Abcam), rotenone (1 μM; 2 days), mevalonate (200 μM; 17 h or 2 days) (MilliporeSigma), BAY-876 (3.3–30.0 μM), everolimus (1 or 5 μM, 19.5 h or 2 days), temsirolimus (1 or 5 μM; 19.5 h or 2 days), shikonin (1 μM; 2 days), PT2385 (10 μM; 19.5 h), TC-S7009 (10 μM; 19.5 h) (Selleck Chemicals), simvastatin (0.5–3.0 μM; 4.5, 6, 14, 17, 21.5, 25 h or 2 days), atorvastatin (1–3 μM; 2 days) and metformin (1–5 mM; 17 h or 2 days) (Tokyo Kasei Kogyo) were reconstituted in dimethyl sulfoxide (DMSO; FUJIFILM Wako Pure Chemical Corporation).

Techniques: Control, Incubation, Western Blot, Expressing, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Isolation, Binding Assay, Knock-Out