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. 2019 May 13;44(1):227–239. doi: 10.3892/ijmm.2019.4195

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Copyright: © Peng et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Effects of NMDA-CM on BLM-treated MLE-12 cells. Normal or 3 mM NMDA-CM was used to co-culture 1 μg/ml BLM-treated MLE-12 cells for 24 h. Furthermore, 10 ng/ml HGF was simultaneously used to treat MLE-12 cells. (A) Protein expression levels of XBP1 and Bip in MLE-12 cells were measured by western blotting. (B and C) Semi-quantitative analysis of XBP1 and Bip protein expression levels. (D) Immunofluorescence staining of Ki67 (red) in MLE-12 cells. DAPI (blue) staining was used to detect the nuclei (magnification, ×400). (E) Semi-quantitative analysis of Ki67-positive MLE-12 cells. (F) Apoptotic cells were identified by Annexin V-fluorescein isothiocyanate/PI staining by flow cytometry. (G) Quantitative analysis of total apoptotic Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ cells is shown. Data are presented as the means ± standard error of the mean, n=3. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. BLM, ^&P<0.05, ^&&P<0.01 vs. BLM + CM, ^$P<0.05, ^$$P<0.01, ^$$$P<0.001 vs. BLM + NMDA-CM. Bip, immunoglobulin heavy chain-binding protein; BLM, bleomycin; CM, conditioned medium; HGF, hepatocyte growth factor; MSC-CM; mesenchymal stromal cell-derived CM; NMDA-CM, NMDA-preconditioned MSC-CM; NMDA, N-methyl-D-aspartate; PI, propidium iodide; XBP1, X-box binding protein 1.

Effects of NMDA-CM on BLM-treated MLE-12 cells. Normal or 3 mM NMDA-CM was used to co-culture 1 μg/ml BLM-treated MLE-12 cells for 24 h. Furthermore, 10 ng/ml HGF was simultaneously used to treat MLE-12 cells. (A) Protein expression levels of XBP1 and Bip in MLE-12 cells were measured by western blotting. (B and C) Semi-quantitative analysis of XBP1 and Bip protein expression levels. (D) Immunofluorescence staining of Ki67 (red) in MLE-12 cells. DAPI (blue) staining was used to detect the nuclei (magnification, ×400). (E) Semi-quantitative analysis of Ki67-positive MLE-12 cells. (F) Apoptotic cells were identified by Annexin V-fluorescein isothiocyanate/PI staining by flow cytometry. (G) Quantitative analysis of total apoptotic Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ cells is shown. Data are presented as the means ± standard error of the mean, n=3. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. BLM, ^&P<0.05, ^&&P<0.01 vs. BLM + CM, ^$P<0.05, ^$$P<0.01, ^$$$P<0.001 vs. BLM + NMDA-CM. Bip, immunoglobulin heavy chain-binding protein; BLM, bleomycin; CM, conditioned medium; HGF, hepatocyte growth factor; MSC-CM; mesenchymal stromal cell-derived CM; NMDA-CM, NMDA-preconditioned MSC-CM; NMDA, N-methyl-D-aspartate; PI, propidium iodide; XBP1, X-box binding protein 1.