Fig 1: SLC25A22 accelerates cell cycle progression and inhibits cell apoptosis of osteosarcoma cells. A, SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells were subjected to cell cycle detection by flow cytometry. The results were analyzed with Modifit software and the proportions for each period were calculated. B, SLC25A22, cyclin B1, cdc25c, and cyclin D1 protein levels were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells. C, Forty-eight hours after U2OS and Saos-2 cells were transfected with shRNA (SLC25A22 KD or control), cells were stained with FITC-Annexin V and PE-PI, and apoptotic cells were detected by flow cytometry and subsequently analyzed with Flowjo software. D, Cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bad were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells.
Fig 2: Cells lacking hTim8a have no defects in the TIM22, TIM23 or carrier biogenesis pathway.(A–E) Mitochondria were isolated from control, (A) hTim9MUT HEK293 cells, (B) hTim8aKO HEK293 cells, (C) hTim8aMUT SH-SY5Y cells, (D) hTim8bKO HEK293 cells, or (E) hTim8bKO SH-SY5Y cells prior to solubilisation in 1% digitonin-containing buffer. Mitochondrial lysates were subjected to Blue-Native electrophoresis prior to immunoblotting using the indicated antibodies. (F–H) [35S]-hTim23, [35S]-hTim22 or [35S]-GC1 were incubated with mitochondria isolated from control and hTim8aKO or hTim8bKO) HEK293 cells for the indicated time in the absence or presence of a mitochondrial membrane potential (??) prior to Proteinase K treatment. Samples were separated by SDS-PAGE or solubilised in 1% digitonin-containing buffer and separated by BN-PAGE and visualised using autoradiography. (I) Mitochondria isolated from control SH-SY5Y and hTim8aMUT SH cells were incubated with [35S]-hTim23 or [35S]-hTim22 for 60 min in the presence or absence of membrane potential (??) before Proteinase K treatment. Mitochondria were reisolated and solubilised in digitonin prior to BN-PAGE and subsequent immunoblotting.
Fig 3: SLC25A22 is highly expressed in human osteosarcoma. A, The total RNA of 40 osteosarcoma tissues and their adjacent tissues was extracted, and SLC25A22 mRNA was detected by RT-PCR. B, The protein of 40 osteosarcoma tissues and their adjacent tissues was extracted and the protein level of SLC25A22 was detected by Western blot. C, The expression of SLC25A22 in osteosarcoma tissues and paracancerous tissues was detected by IHC. D, A typical IHC schematic showing the expression of SLC25A22 in osteosarcoma tissues and adjacent tissues. E, Survival curve of patients with osteosarcoma expressing SLC25A22 at high and low levels. IHC indicates immunohistochemical; RT-PCR, Reverse transcriptase-polymerase chain reaction.
Fig 4: SLC25A22 increases proliferation of osteosarcoma cell lines in nude mice models. A, 293, SF-86, U2OS, Saos-2, 143B, MG63, HOS cells were lysed and SLC25A22 protein levels were detected by Western blot. B, SLC25A22 knockdown efficiency and overexpression were detected by Western blot. C, The knockdown and overexpression efficiency of SLC25A22 was examined by RT-PCR. D, SLC25A22 KD U2OS and Saos-2 cells and overexpressing HOS cells were cultured in 96-well plates, and the number and viability of the cells were respectively measured using CCK-8 after 1, 2, and 3 days of culture. E, SLC25A22 KD U2OS and Saos-2 cells and overexpressing HOS cells were subjected to colony formation experiments, and the number of clones was recorded. F, Saos-2 cells and HOS cells were infected with lentivirus containing SLC25A22-shRNA and SLC25A22 overexpression vector, respectively. Stably infected Saos-2 cells and HOS cells were implanted subcutaneously in nude mice. After 3 weeks, the tumor was removed and photographed. G, During tumor growth, the tumor volume was recorded. H, At the end of the experiment, the tumor weight of each group was measured. I, After the above-mentioned grouped tumor tissues were embedded in paraffin, the expression of PCNA was detected by immunohistochemistry. J, The tumor paraffin sections were subjected to the TUNEL assay, and green fluorescence represented tissue cells with DNA damage.
Fig 5: A, SLC25A22 promotes the invasion and metastasis of osteosarcoma cells by altering the PTEN signaling pathway. Wound healing assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in 6-well plates for wound healing experiments, and healing was observed and photographed at 24 hours. B, Cell invasion assay: stable SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells were plated in Transwells and the invaded cells were stained with crystal violet and photographed. C, E-cadherin, vimentin, and MMP-9 protein levels were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. D, PTEN, phosphorylated Akt, and phosphorylated FAK were detected by Western blot in SLC25A22 knockdown U2OS, Saos-2 cells, and overexpressed HOS cells. E, Control and SLC25A22 knockdown Saos-2 cells were used to construct a lung metastasis model, and HE staining showed 2 groups of lung metastases. F, Comparison of the number of lung metastatic cells in the control and SLC25A22 knockdown groups. G, The number of mice with lung metastases in the two groups, control and SLC25A22 knockdown lung metastasis model mice.
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