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Supplementary Material 1

Additional file 1: Figure S1. (A) The expression of ARPs among mesenchymal (MES), proneural (PN), and classical (CL) phenotypes in TCGA GBM. (B) Kaplan–Meier curves visualizing the overall survival of CCGA-GBM patients stratified according to expression of ARPs. (C) The expression of ARPs among MES, PN, and CL phenotypes in CGGA GBM. (D) GSEA exhibited a positive correlation between ARPC1B expression and MES phenotypes, and a negative correlation with PN phenotypes. (E) Single-cell RNA sequencing of GSE138794 visualizing the expression of ARPs other than ARPC1B. Fig. S2. (A) Correlation analysis of ARPC1B with CD44, YKL-40, OLIG2 and SOX2 in TCGA-GBM and CGGA-GBM, respectively. (B) Western blot analysis of ARPC1B and SOX2 protein levels in GSC 8-11 overexpressing ARCP1B. (C, D) Representative images and quantification of tumor sphere formation of GSC 8-11 (C) and GSC 11 (D) transduced with vector or ARPC1B. Scale bar, 100μm. (E) The protein expression of ARPC1B in GSC 20 and GSC 267 under different IR dose treatments. Fig. S3. (A) Flow cytometric analysis showing the effect of ARPC1B overexpression on the apoptosis in IR-treated (6Gy) GSC 8-11 cells. The right panels showing the quantification of apoptosis rate. (B) Representative images and quantification of comet assay showing the effect of ARPC1B overexpression on DNA damage of GSC 8-11 with IR treatment (6 Gy). Scale bar, 20μm. (C) Representative images and quantification of γ-H2AX IF staining showing the effect of ARPC1B knockdown on DNA damage of GSC 267 and GSC 20 with IR treatment (6 Gy). Scale bar, 40μm. (D) Representative images and quantification of γ-H2AX IF staining in GSC 8-11. Scale bar, 40μm. (E) Cell-cycle analysis of GSC 267, GSC 20 and GSC 8-11 in different treatment groups. The proportions of cells arrested in G2/M phase were quantified (right panel). Fig. S4. (A, B) Bioluminescence imaging of tumor size on day 7 in sh-control, sh-ARPC1B#1 and sh-ARPC1B#2 GSC 267 (A) or GSC 20 (B) xenograft nude mice in indicated groups. The right panel shows the quantification of photon counts of GSC 267 and GSC 20 xenografts. (C, D) Bioluminescence imaging of tumor size on day 7 (C) and day 30 (D) in Vector or ARPC1B-transfected GSC 8-11 xenograft nude mice receiving or exempt from IR treatment. The right panel showing the quantification of photon counts of GSC 8-11 xenografts. Fig. S5. (A) Representative images and quantification of IHC staining for CD44 in sections of non-IR GSC 267 xenografts (upper, scale bar, 200μm), and TUNEL staining in sections of IR treated GSC 267 xenografts (lower, scale bar, 200μm). (B) Representative images and quantification of IHC staining for CD44 in sections of non-IR GSC 8-11 xenografts (upper, scale bar, 200μm), and TUNEL staining in sections of IR treated GSC 8-11 xenografts (lower, scale bar, 200μm). (C) Representative images of H&E staining in sections from indicated xenografts. Scale bar, 400μm. (D) Western blotting analysis of protein levels of DBN1, ACTN4, FLNA, and CORO1C upon knockdown of ARPC1B in GSC 20 and GSC 267, or overexpression of ARPC1B in GSC 8-11. Fig. S6. (A) Co-IF staining exhibiting the distribution of ARPC1B with IFI16 or HuR in GSC 20. Scale bar, 5μm. (B) The protein levels of IFI16 and HuR after overexpression of ARPC1B in GSC 8-11. (C) The mRNA expression of IFI16 and HuR assessed by qRT-PCR assay. (D) The protein levels of IFI16 and HuR in sh-control or sh-ARPC1B-GSCs treated with 100μg/ml CHX for indicated times. (E) Representative images and quantification of IHC staining for ARPC1B, HuR and IFI16 in different groups of GSC 267 xenograft sections (scale bar, 200μm). Fig. S7. (A) Western blotting analysis showing the effect of TRIM21 knockdown on the protein levels of IFI16 and HuR in sh-ARPC1B GSCs. (B) Western blotting analysis showing the effect of ARPC1B knockdown in GSC 20 and GSC 267, or ARPC1B overexpression in GSC 8-11 on the protein levels of STAT3, p-STAT3, P65, and p-P65. (C) Western blotting analysis of protein levels of ARPC1B, IFI16, HuR, P65, p-P65, STAT3, p-STAT3 and, CD44 in GSC 20 treated with indicated interventions. (D) Western blotting analysis showing that knockdown of TRIM21 could reverse the effect of ARPC1B inhibition on IFI16 and HuR ubiquitination. GSCs were pretreated with MG132 (10 µM) for 6 hours before cell lysates were collected. Fig. S8. (A) Representative images of tumor sphere formation of GSC 267 and GSC 20 treated with indicated interventions. Scale bar, 100μm. (B) Representative images of flow cytometry assays showing apoptosis of GSC 267 and GSC 20 treated with indicated interventions. (C) Representative images of comet assays showing DNA damage of GSC 267 and GSC 20 treated with indicated interventions. Scale bar, 20μm. Fig. S9. (A) The correlation between the ARPC1B expression and drug sensitivity assessed by Spearman algorithm. (B) CCK-8 assay in GSC 267 and GSC 20 treated with different concentrations of AZD6738 for 48 h. (C) Representative images and quantification of apoptosis assays (upper panel) and comet assays (lower panel, scale bar, 20μm) for GSC 20 and GSC 267 upon treatment with the indicated interventions. (D) The representative images and quantification of apoptosis assays (upper panel) and comet assays (lower panel, scale bar, 20μm) for GSC 8-11 upon treatment with the indicated interventions. The right panels are the quantification of apoptosis rate and DNA damage, respectively. (E) Representative images and quantification of TUNEL staining in sections of GSC 267 xenografts for different groups. Scale bar, 200μm. (F) Bioluminescence imaging of tumor size on day 7 in GSC 267 xenograft nude mice treated with the indicated interventions. (G) The quantification of photon counts on day 7 of the GSC 267 xenografts. Supplementary Materials and Methods.

Additional file 1: Supplementary Figure 1. A The IC50 for TMZ and expression of PDIA3P1 of 10 glioma cell lines (TMZ sensitive and TMZ resistant cell lines). B Patients with high PDIA3P1 expression exhibited shorten progress disease survival time. C The PDIA3P1 expression in 4 glioblastoma cell lines. D PDIA31P was knocked down in U118MG and U87MG cells by two different shRNA and overexpressed in LN229 and U251 cells. E Cell proliferation was assessed by CCK-8 assay. Supplementary Figure 2. A B Bioluminescence imaging of tumor growth on day five in U118MG (A) and U251 (B) xenograft nude mice. C D Kaplan–Meier visualized survival time for animals in different groups for U118MG (C) and U251 (D). E F Representative images of hematoxylin and eosin (H&E) staining in sections from U118MG (E) and U251 (F) xenografts. Scale bar, 400 μm. Supplementary Figure 3. A Patients were divided into high and low PDIA3P1 expression groups according to PDIA3P1 expression, and GSVA analysis was performed, and the results were presented using heatmap. B Heatmap visualized immune infiltration using ssGSEA, and the correlation between PDIA3P1 expression and immune infiltration was analyzed using the chi-square test (lower panel). C Waterfall plot showing tumor somatic cell mutations in low (left panel) and high (right panel) PDIA3P1 expression groups. Supplementary Figure 4. A B DNA damage was assessed by comet (A. Scale bar, 100 μm) and γ-H2AX IF staining (B. Scale bar, 10 μm) assays. Knockdown of PDIA3P1 significantly promoted TMZ treatment-induced DNA damage. C D Cell proliferation was assessed by EdU (C. Scale bar, 200 μm) and colony formation (D. Scale bar, 200 μm) assays. Knockdown of PDIA3P1 further significantly increased the proliferation inhibitory effect caused by TMZ. The lower panel exhibited the quantification of EdU and colony formation assays. E F Representative images of comet (E. Scale bar, 100 μm) and γ-H2AX IF staining (F. Scale bar, 10 μm) assays for LN229 cells. Overexpression of PDIA3P1 remarkedly reduced TMZ treatment-induced DNA damage. Supplementary Figure 5. A B DNA damage was assessed by γ-H2AX IF staining assay in GSC20. Knockdown of PDIA3P1 significantly promoted TMZ treatment-induced DNA damage, whereas overexpression of PDIA3P1 counteracted the DNA damage induced by TMZ treatment (Scale bar, 10 μm). C D DNA damage was assessed by comet assay in GSC20 (Scale bar, 100 μm). E F Cell proliferation was assessed by EdU assay in GSC20. Knockdown of PDIA3P1 promoted proliferation inhibition caused by TMZ treatment, whereas overexpression of PDIA3P1 partially counteracted TMZ-mediated cell growth inhibition (Scale bar, 200 μm). Supplementary Figure 6. A The expression of PDIA3P1 in classical, proneural (PN) and mesenchymal (MES) tissues in the CCGA dataset. B ROC curves of PDIA3P1 for MES-GBM subtype prediction in CCGA. C Knockdown of PDIA3P1 in GSC20 and GSC267, overexpression of PDIA3P1 in GSC8–11. D Neurospheres formation assay revealed knockdown of PDIA3P1 reduced self-renewal capacity of GSC20 and GSC267. Scale bar, 200 μm. The right panels were the quantification of sphere diameters. E The quantification of IF staining for SOX2 and CD44 in GSC20 (left panel) and GSC267 (right panel). Knockdown of PDIA3P1 resulted downregulation of CD44 and upregulation of SOX2 in GSC20 and GSC267. Supplementary Figure 7. A B Representative images of γ-H2AX IF staining (A. Scale bar, 10 μm) and comet (B. Scale bar, 200 μm) assays revealed DNA damage in GSC267. C D Representative images of γ-H2AX IF staining (C. Scale bar, 10 μm) and comet (D. Scale bar, 200 μm) assays revealed DNA damage in GSC8–11. E The expression of C/EBPβ mRNA detected by qPCR. F Western blotting analysis of C/EBPβ in GSC8–11 PDIA3P1 stable overexpressed and control cells after treatment with CHX (100 μg/ml) for indicated times. G Western blotting analysis of p-P38 and P38 expression with increasing concentrations of TMZ treatment. Supplementary Figure 8. A Visualization of the Fa-CI (fraction afected Combination Index) results obtained form CompuSyn for U118MG, GSC20, GSC267, and U251. B The detailed sequence of PDIA3P1 promoter (610–1310) and three predicted binding sites for JUN. Supplementary Figure 9. A B EdU assay (A. Scale bar, 200 μm) and apoptosis assay (B) showed that TMZ combined with NEF exhibited excellent anti-tumor cells effects, respectively. C Bioluminescence imaging of tumor growth on day five in GSC267 xenograft nude mice. D Kaplan–Meier visualized survival time for mice in different treatment groups. E Representative images of hematoxylin and eosin (H&E) staining in sections from GSC267 xenografts. Scale bar, 400 μm.

Additional file 2: Figure S1. Functional analysis of positive genes correlated with circNEIL3 in our local glioma tissues. A KEGG pathway analysis of the positive regulated genes with circNEIL3 in our local glioma dataset. B GSEA analysis revealed that genes with high circNEIL3 expression group were enriched for pathways of malignant tumors in our local glioma dataset. C GSVA enrichment analysis showing the activation status of biological pathways in the circNEIL3-high and circNEIL3-low groups. The heatmap was used to visualize these biological processes. Yellow represents activated pathways, black represents moderately activated pathways, and blue represents inhibited pathways. Figure S2. circNEIL3 promotes gliomagenesis in vitro. qRT-PCR assays showing the expression of (left) circNEIL3 and mRNA NEIl3(right) in GBM cells and GSCs transfected with (A) si-NC or si-circNEIL3, and (B) ov-NC or ov-circNEIL3, n = 3. Representative EDU assay images showing the proliferation ability of GBM cells transfected with (C) si-NC or si-circNEIL3, and (D) ov-NC or ov-circNEIL3. Quantification histogram represented relative cell numbers, n = 3, scale bar, 200um. Representative colony formation assays showing the proliferation ability of GBM cells transfected with (E) si-NC or si-circNEIL3, and (F) ov-NC or ov-circNEIL3. Quantification histogram represented relative cell numbers, n = 3. G Representative images of tumor spheres formation assays showing the tumor spheres formation ability of GSCs transfected with (G) si-NC or si-circNEIL3, and (H) ov-NC or ov-circNEIL3. Quantification histogram represented average sphere dimeter, n = 3, scale bar, 200um. limiting dilution assay showing the self-renewal ability of GSCs transfected with (I) si-NC or si-circNEIL3, and (J) ov-NC or ov-circNEIL3. K Represented H&E staining images for a subgroup of animal sacrificed simultaneously in each group, n = 5 for each group, scale bar, 10 ��m. All data are presented as the means �� SD, *P 0.05, *P