Additional file 1 of SH3GLB1-related autophagy mediates mitochondrial metabolism to acquire resistance against temozolomide in glioblastoma
Creators: name: Chien, Chia-Hung
name: Yang, Wen-Bin
name: Chuang, Jian-Ying
name: Lee, Jung-Shun
name: Liao, Wei-An
name: Huang, Chih-Yuan
name: Chen, Pin-Yuan
name: Wu, An-Chih
name: Yang, Shun-Tai
name: Lai, Chien-Cheng
name: Chi, Pei-I
name: Chu, Jui-Mei
name: Cheng, Siao Muk
name: Liu, Chan-Chuan
name: Hwang, Daw-Yang
name: Chen, Shang-Hung
name: Chang, Kwang-Yu
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. (A) Differentiation of mitochondria-related genes in A172 TMZ resistance cells (A172-R) with shSOD2 or shControl is shown in the heatmap graphs. A total of 84 genes was assessible from the assay. Among them, however, only 71 of the items were found in the database of mitochondria-related genes according to MitoCarta 2.0. (B) The correlation between SH3GLB1 and SOD2 was also shown in TCGA-GBM dataset and the 14 paired patients. (C) Nine cases showing expression of SH3GLB1 in the paired primary- and recurrent-tissues using IHC staining (100x and 400x magnification). (D) Kaplan-Meier curves of TCGA-GBMLGG (GBM and low-grade glioma) database showed higher SH3GLB1 levels caused poor survival. Figure S2. Ingenuity Pathway Analysis was applied for analysis of the major mitochondria-related molecular and cellular functional alterations in the transcriptome data from 14 RNA-seq data from paired recurrent and treatment-naïve high-grade glioma samples. As shown in the figure, sample numbers of down- (left column) or up-regulated (right column) genes. In each grid, the color is determined by the rank percentile, and the number represents sample that fulfills the criteria. The levels and significance of the genes ratio from CGGA glioma database (recurrent versus primary) are also shown aside. Note that none of the genes in complex II had CGGA database significance better than **, and the genes in complex V and CoQ synthesis was not higher in the resistant groups in the 14 paired samples. ***p &lt; 0.001, **p &lt; 0.01, *p &lt; 0.05, NS: not significant. NA: data not available. Figure S3. (A) Five naïve glioblastoma tumor samples were used for single-cell transcriptome and were sorted into nine clusters according to their gene expression (Fig. 2A). The heatmap graph shows the common related genes of GBM and tumor-initiating cells among the clusters. Note that clusters 3, 7, 8, and 9 are identified as immunocytes because of the presence of the markers such as CD3E/G, GZMA, ITGAM, ITGAX. (B) The clusters reclassified by the studies of Neftel et al. (Cell, 2019) were divided into four cellular states. Figure S4. (A) Cell metabolic phenotype profile were measured using Seahorse Analyzer (OCR and ECAR). (B) The individual OCR parameters were measured using the indicated reagents and plotted in Fig. 5B, with the statistic graphs showing the parameters of maximal respiration, spare respiratory capacity, and nonmitochondrial respiration. (C) The parental cells transfected with SH3GLB1-overexpressing vector were stained with a JC-1 dye, showing the enhanced fluorescent ratio. Figure S5. (A) IACS-010759 effect to cell density for 72 hours of the parental cells with or without SH3GLB1 overexpression are shown in the bar graph. (B) IACS-010759 effect to cell density for 72 hours of the resistant cells with or without SH3GLB1 knockdown are shown in the bar graph. (*P &lt; 0.05) Figure S6. (A) The U87MG-R luciferase-carrying cells (U87MG-R-luc-EGFP) were transfected with shSH3GLB1 used in animal experiment. SOD2 levels did not be affected by SH3GLB1 deficiency. (B) The tumor volume was measured at Day 20 and Day 23 according to the National Cancer Institute formula. (C) The body weight was recorded at the indicated day as the statistic graph. (*P &lt; 0.05) (D) The image shows the tumor extracted from euthanized mice (scale bar = 1 cm) (E) The SH3GLB1 expression in the tissue using an IHC staining is shown (scale bar = 200 μm). (F) The body weight was recorded and shown in the statistical graph. Figure S7. Kaplan-Meier curves of GBM database from CGGA showed the survival of the indicated subunits genes of OXPHOS complex. Figure S8. A172 or A172-R cells are used in the results including roles of SH3GLB1 on Sp1 promoter, Sp1 expression, 36 autophagy levels, OXPHOS levels, cell density and OCR analysis. Figure S9. Pt#5 or Pt#5-R cells are used in the results including roles of SH3GLB1 on Sp1 promoter, Sp1 expression, autophagy levels, OXPHOS levels, cell density and OCR analysis.