Fig 1: Cytoprotective effect of CYGB. (A) Knockdown of CYGB increased basal intracellular ROS levels. (B) G361-shCTR and G361-shCYGB cells were treated with 7.5 µM RSL3 and cell viability was measured after 24 h using propidium iodide (PI) staining. (C) The average calibrated normalized relative quantities (CNRQ) of HO-1 and NRF2 mRNA under basal conditions (UT) or upon RSL3 treatment. CNRQ values were normalized to B2M and YWHAZ. (D) Immunoblotting results of NRF2, HO-1 and CYGB 6h after RSL3 treatment. ACTB was used as loading control. (E) The average fold change in protein expression of HO-1, NRF2 and CYGB in G361-shCTR and G361-shCYGB cells, compared to the untreated G361-shCYGB samples (set as 1). Measured immunoblot signals were normalized to the loading control ACTB. (F) Average ratio of the measured red (reduced) over green (oxidized) signal of the BODIPY 581/591 C11 reagent in G361-shCTR and G361-shCYGB cells. Cells were either untreated or treated with 7.5 µM RSL3 or 100 µM cumene hydroperoxide (positive control). All results are depicted as the mean with S.E.M. of three independent experiments (n = 3). (A) Student’s t-test (** p = 0.01), (B–F) two-way ANOVA (*/# p = 0.05; **/## p = 0.01; *** p = 0.001; ****/#### p = 0.0001, ns: non-significant).
Fig 2: Intracellular ROS levels are elevated. Intracellular ROS levels were determined with the Incucyte, using the CellROX Green reagent. (A) The basal ROS level of all used cell lines is shown as the average green object mean intensity. (B) Average CYGB protein expression in transduced G361 and A375 cells. G361-shCYGB and A375-hCYGB protein levels were normalized to their respective transgenic controls. B2M was used as loading control. (C) 5min pPBS treatment of A375 cells and (D) 7 min pPBS treatment of G361 cells induced a significant increase in green fluorescent signal compared to the untreated control. (E & F) Intracellular ROS levels of CYGB overexpressing (A375-hCYGB) and CYGB knockdown (G361-shCYGB) cells compared to control cell lines A375-GUS and G361-shCTR. Pre-treatment with 1 mM NAC (pPBS + NAC) significantly reduced intracellular ROS levels compared to the control (mean ± S.E.M; n = 3). Asterisks represent significant difference compared to the respective control cell lines. Two-way ANOVA (**p = 0.01; ****p = 0.0001; ##p = 0.01; ###p = 0.001; ####p = 0.0001). UT, untreated; pPBS, plasma-treated PBS; NAC, N-acetyl cysteine. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 3: Basal mRNA expression levels. Average mRNA expression levels of CYGB, HO-1, and NRF2 in A375-GUS, A375-hCYGB cells, G361-shCTR, and G361-shCYGB. Calibrated normalized relative quantities were normalized to B2M and YWHAZ (mean ± S.E.M; n = 3). Individual values of replicates are depicted as black dots, squares, clubs, or triangles. One-way ANOVA (****p = 0.0001).
Fig 4: Basal CYGB-dependent transcriptome. Comparison of the G361-shCTR and G361-shCYGB transcriptomes under basal conditions. (A) Enhanced volcano plot plots the differentially expressed genes based on their Log2 fold change (Log2FC) and -Log10 p-adjusted value (-Log10 P). CYGB was excluded from plotting. Individual genes were color coded depending on the set Log2FC and (-Log10 P) thresholds. (B) Fold change mRNA expression compared to G361-shCYGB cell line (set as 1). Relative quantities were normalized to B2M and YWHAZ (mean ± S.E.M; n = 3). Individual values of replicates are depicted as red dots or blue triangles. Student's t-test (**p = 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 5: Beta-2 microglobulin (B2M) is a potential marker for the prognosis of glioma patients. A. Survival probabilities of glioma patients in the high and low B2M groups across three databases. B. Forest plot shows that high B2M expression corresponded to a poor survival time compared to that of low B2M expression in glioma patients from three databases
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