Journal: EMBO Molecular Medicine

Article Title: Targeting cIAP2 in a novel senolytic strategy prevents glioblastoma recurrence after radiotherapy

doi: 10.1038/s44321-025-00201-x

Figure Lengend Snippet: ( A ) Representative images of SA-β-gal staining of GBM cell lines mock-irradiated (Mock) or irradiated (IR) with 10 Gy of X-rays and then allowed to recover for 10 days ( n = 3 with at least 100 nuclei scored for each replicate). Nuclei are stained with DAPI (blue). Plots show mean percentage of SA-β-gal-positive cells +/− SD. A two-tailed Student’s t test was performed; LN229 P = 0.000009, A172 P = 0.000027, U118 P = 0.000141, U87 P = 0.007801. Scale bar, 100 μm. ( B ) Whole cell extracts from mock-irradiated or irradiated GBM cell lines were western blotted with anti-p21 antibody. Actin serves as loading control. ( C ) Volcano plot representation of differentially expressed genes from mock-irradiated vs . irradiated LN229 or A172 cells ( n = 3), as assessed by RNA sequencing 10 days after treatment with 10 Gy of X-rays. Differential gene expression analysis was performed using edgeR. Gene-specific dispersions were estimated using a tagwise dispersion model and statistical significance was assessed using the likelihood ratio test (LRT). The false discovery rate (FDR) was calculated using the Benjamini–Hochberg procedure. Genes with an FDR < 0.05 with log 2 fold change (Log 2 FC) cutoff of −1 and 1 were considered differentially expressed, as denoted by dashed lines. Core SASP genes that were further validated by qRT-PCR are represented by numbers. ( D ) Total RNA was isolated from GBM cells 10 days after irradiation with 10 Gy of X-rays or from mock-irradiated cells and expression of SASP genes assessed by qRT-PCR ( n = 3 biological replicates comprising three technical replicates each). Plots show mean fold change (Log 2 FC) in gene expression +/− SD of SASP-related genes in irradiated GBM cells relative to mock-irradiated cells. A two-tailed Student’s t test was performed; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (please refer to Appendix Table for the exact p values). ( E ) Conditioned media were collected from GBM cells 10 days after irradiation with 10 Gy of X-rays ( n = 3 biological replicates comprising three technical replicates each), and cytokine levels were measured by multiplex bead-based immunoassay. Plots show mean fold change (Log 2 FC) in cytokine levels +/− SD in media from irradiated cells relative to mock-irradiated cells. A two-tailed Student’s t test was performed; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (please refer to Appendix Table for the exact P values). .

Article Snippet: U118 , ATCC , HTB-15.

Techniques: Staining, Irradiation, Two Tailed Test, Western Blot, Control, RNA Sequencing, Gene Expression, Dispersion, Quantitative RT-PCR, Isolation, Expressing, Multiplex Assay, Bead-based Assay

Journal: EMBO Molecular Medicine

Article Title: Targeting cIAP2 in a novel senolytic strategy prevents glioblastoma recurrence after radiotherapy

doi: 10.1038/s44321-025-00201-x

Figure Lengend Snippet: ( A ) Representative images of GBM cell lines immunofluorescence stained for Lamin B1 (green) and ( B ) Ki67 (green), 10 days after irradiation (IR) with 10 Gy of X-rays or mock-irradiation (Mock). n = 3 with at least 100 nuclei scored for each replicate. Nuclei are stained with DAPI (blue). Plots show mean percentages +/− SD of Lamin B1- or Ki67-positive cells. A two-tailed Student’s t test was performed; Lamin B1 - LN229 P = 0.00000163109, A172 P = 0.00000004724, U118 P = 0.00000000002, U87 P = 0.00000702223; Ki67 - LN229 P = 0.000030, A172 p = 0.000002, U118 p = 0.000028, U87 P = 0.000005. Scale bar, 50 μm. ( C ) Heatmap of top 100 differentially expressed genes in LN229 or A172 GBM cells mock-irradiated (Mock) or irradiated (IR) with 10 Gy of X-rays and then allowed to recover for 10 days ( n = 3), as assessed by RNA sequencing. ( D ) Senescence scores of mock-irradiated or irradiated LN229 and A172 cells ( n = 3) generated by analysis of RNA-seq datasets using the SenCan Classifier tool. Score ranges from 0 (no senescence) to 1 (senescence). Plot shows mean senescence score +/− SD for both GBM cell lines. A two-tailed Student’s t test was performed; LN229 P = 0.00002368169637, A172 p = 0.00000000000002. ( E ) Genes involved in SASP, Cytokine-cytokine receptor interaction, JAK-STAT signaling, and NF-kB signaling are significantly enriched in irradiated cells compared to mock-irradiated cells. Normalized Enrichment Scores (NES) are shown in the figure; P = 0. ( F ) SASP-transcription factor network generated by the list of 24 common cytokines secreted by all four senescent GBM cell lines cross-referenced against the TRRUST database. The visualization was generated using Cytoscape version 3.9.1. Transcription factors are shown in green. The cytokines not shown had no available information in the database. .

Article Snippet: U118 , ATCC , HTB-15.

Techniques: Immunofluorescence, Staining, Irradiation, Two Tailed Test, RNA Sequencing, Generated

Journal: EMBO Molecular Medicine

Article Title: Targeting cIAP2 in a novel senolytic strategy prevents glioblastoma recurrence after radiotherapy

doi: 10.1038/s44321-025-00201-x

Figure Lengend Snippet: ( A ) Naive GBM cells were exposed to conditioned media from senescent (CM-SEN) or non-senescent (CM-NS) GBM cells for the indicated times, and activation of the JAK-STAT3 pathway assessed by western blotting with anti-phopho-STAT3 (Y705) antibody. Actin serves as loading control. Recipient cells were serum starved (ss) before addition of CM. ( B ) Phosphorylation of STAT3 after exposure of naive cells to CM-SEN for 30 min was assessed by western blotting after either neutralization of IL-6 in the conditioned media with an anti-IL-6 antibody (N-α-IL-6) or treatment of recipient GBM cells with the JAK inhibitor ruxolitinib, as indicated. ( C ) Serum starved (ss) GBM cells were pulsed with BrdU after exposure to CM-NS, CM-SEN, or CM-SEN in the presence of ruxolitinib (CM-SEN+Ruxo), and immunofluorescence stained with anti-BrdU antibody (red), as indicated ( n = 3 with at least 100 nuclei scored for each replicate). Nuclei are stained with DAPI (blue). Plots show mean percentages of BrdU-positive cells +/− SD. A two-tailed Student’s t test was performed; LN229 P = 0.00183, 0.0028; A172 P = 0.01061, 0.03826; U118 P = 0.00736, 0.02480; U87 P = 0.00002, 0.00002, respectively. Scale bar, 10 μm. .

Article Snippet: U118 , ATCC , HTB-15.

Techniques: Activation Assay, Western Blot, Control, Phospho-proteomics, Neutralization, Immunofluorescence, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Targeting cIAP2 in a novel senolytic strategy prevents glioblastoma recurrence after radiotherapy

doi: 10.1038/s44321-025-00201-x

Figure Lengend Snippet: ( A ) Kaplan–Meier curve showing lack of correlation of Bcl2l10 expression levels with prognosis in GBMLGG patients in TCGA ( n = 606) and CGGA ( n = 657) cohorts as evidenced by Hazard Ratio (logrank) of 0.9692 and 0.8629 and P values of 0.1943 and 0.2640, respectively. ( B ) Plot shows mean relative expression of BIRC2 + /− SD in mock-irradiated vs. irradiated (IR) GBM cells 10 days after exposure to 10 Gy of X-rays, as assessed by qRT-PCR ( n = 3 biological replicates comprising 3 technical replicates each). A two-tailed Student’s t test was performed; exact P values from left to right: 0.0000001, 0.0010112, 0.0044335, 0.9295618. ( C ) Whole cell extracts from mock-irradiated or irradiated GBM cell lines were western blotted with anti-cIAP1 antibody. Actin serves as loading control. ( D ) Senescent GBM cells (10 days after exposure to 10 Gy) were treated with the IKK inhibitor BMS-345541 (BMS) or DMSO as control for 72 h ( n = 3 biological replicates comprising 3 technical replicates each), and mean relative expression of BIRC3 + /− SD was assessed by qRT-PCR (a two-tailed Student’s t test was performed; A172 p = 0.00000003, 0.00000232; U118 p = 0.00000220, 0.00026887; U87 P = 0.00000818, 0.00118914, respectively) or ( E ) western blotting for cIAP2. ( F ) Naive GBM cells were exposed to conditioned media from senescent (CM-SEN) or non-senescent (CM-NS) cells for the indicated times, and expression of cIAP2 assessed by western blotting. ( G ) Naive GBM cells were treated with BMS-345541 or DMSO as control for 2 h before exposure to CM-SEN ( n = 3 biological replicates comprising 3 technical replicates each), and mean relative expression of BIRC3 + /− SD was assessed by qRT-PCR (a two-tailed Student’s t test was performed; A172 P = 0.000627, 0.000622; U118 P = 0.000001, 0.000001; U87 P = 0.000593, 0.000936, respectively) or ( H ) western blotting for cIAP2. ( I ) GBM cells were exposed to conditioned media from senescent or non-senescent cells ( n = 3) and radiation sensitivity measured by the colony survival assay. The mean percentage of surviving colonies +/− SD ( y axis) is plotted against the corresponding radiation dose ( x axis). .

Article Snippet: U118 , ATCC , HTB-15.

Techniques: Expressing, Irradiation, Quantitative RT-PCR, Two Tailed Test, Western Blot, Control, Clonogenic Cell Survival Assay

Journal: EMBO Molecular Medicine

Article Title: Targeting cIAP2 in a novel senolytic strategy prevents glioblastoma recurrence after radiotherapy

doi: 10.1038/s44321-025-00201-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: U118 , ATCC , HTB-15.

Techniques: Biomarker Discovery, Purification, Sequencing, Negative Control, Plasmid Preparation, Fluorsave, SYBR Green Assay, Western Blot, Stripping, Software

Journal: EMBO Molecular Medicine

Article Title: Targeting cIAP2 in a novel senolytic strategy prevents glioblastoma recurrence after radiotherapy

doi: 10.1038/s44321-025-00201-x

Figure Lengend Snippet: ( A ) List of BCL-2 and IAP family members showing fold changes (Log 2 FC) in gene expression in irradiated (IR) LN229 or A172 cells relative to mock-irradiated (mock) cells along with mRNA transcripts levels in counts per million (CPM), as assessed by RNA sequencing 10 days after irradiation with 10 Gy of X-rays ( n = 3). Genes with Log 2 fold change greater than 3 are highlighted in red. ( B ) MA plot showing abundance of transcripts represented by Log 2 CPM ( y axis) vs . Log 2 fold change in gene expression ( x axis) in irradiated cells relative to mock-irradiated cells ( n = 3 biological replicates per condition for each cell line). BCL-2 and IAP family gene members are denoted with numbers. Genes with log 2 fold change (Log 2 FC) cutoff of −1 and 1, and Log 2 CPM higher than −1 were considered differentially expressed, as denoted by dashed lines. ( C ) Kaplan–Meier curve showing correlation of higher BIRC3 expression levels with poor prognosis in GBMLGG patients in TCGA ( n = 606) and CGGA ( n = 657) cohorts as evidenced by Hazard Ratio (logrank) of 3.441 and 1.811, respectively. P < 0.0001 for both plots. ( D ) Plot shows mean relative expression of BIRC3 + /− SD in mock-irradiated vs. irradiated (IR) GBM cells 10 days after exposure to 10 Gy of X-rays, as assessed by qRT-PCR ( n = 3 biological replicates comprising three technical replicates). A two-tailed Student’s t test was performed; exact P values from left to right: 0.00001, 0.00048, 0.00397, 0.00012. ( E ) Whole cell extracts from mock-irradiated or irradiated GBM cell lines were western blotted with anti-cIAP2 antibody. Actin serves as loading control. ( F ) Senescent LN229 cells (10 days after exposure to 10 Gy) were treated with the IKK inhibitor BMS-345541 (BMS) or DMSO as control for 72 h ( n = 3 biological replicates comprising three technical replicates), and mean relative expression of BIRC3 + /− SD was assessed by qRT-PCR (a two-tailed Student’s t test was performed; P = 0.00000002, 0.00000003, respectively) or ( G ) western blotting for cIAP2. ( H ) Naive LN229 cells were exposed to conditioned media from senescent (CM-SEN) or non-senescent (CM-NS) cells for the indicated times, and expression of cIAP2 assessed by western blotting. ( I ) Naive LN229 cells were treated with BMS-345541 or DMSO as control for 2 h before exposure to CM-SEN ( n = 3 biological replicates comprising 3 technical replicates) and mean relative expression of BIRC3 + /− SD was assessed by qRT-PCR (a two-tailed Student’s t test was performed; P = 0.00001, 0.00002, respectively) or ( J ) western blotting for cIAP2. .

Article Snippet: BMS-345541 , MedChemExpress , HY-10519.

Techniques: Gene Expression, Irradiation, RNA Sequencing, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Control

Journal: EMBO Molecular Medicine

Article Title: Targeting cIAP2 in a novel senolytic strategy prevents glioblastoma recurrence after radiotherapy

doi: 10.1038/s44321-025-00201-x

Figure Lengend Snippet: ( A ) Kaplan–Meier curve showing lack of correlation of Bcl2l10 expression levels with prognosis in GBMLGG patients in TCGA ( n = 606) and CGGA ( n = 657) cohorts as evidenced by Hazard Ratio (logrank) of 0.9692 and 0.8629 and P values of 0.1943 and 0.2640, respectively. ( B ) Plot shows mean relative expression of BIRC2 + /− SD in mock-irradiated vs. irradiated (IR) GBM cells 10 days after exposure to 10 Gy of X-rays, as assessed by qRT-PCR ( n = 3 biological replicates comprising 3 technical replicates each). A two-tailed Student’s t test was performed; exact P values from left to right: 0.0000001, 0.0010112, 0.0044335, 0.9295618. ( C ) Whole cell extracts from mock-irradiated or irradiated GBM cell lines were western blotted with anti-cIAP1 antibody. Actin serves as loading control. ( D ) Senescent GBM cells (10 days after exposure to 10 Gy) were treated with the IKK inhibitor BMS-345541 (BMS) or DMSO as control for 72 h ( n = 3 biological replicates comprising 3 technical replicates each), and mean relative expression of BIRC3 + /− SD was assessed by qRT-PCR (a two-tailed Student’s t test was performed; A172 p = 0.00000003, 0.00000232; U118 p = 0.00000220, 0.00026887; U87 P = 0.00000818, 0.00118914, respectively) or ( E ) western blotting for cIAP2. ( F ) Naive GBM cells were exposed to conditioned media from senescent (CM-SEN) or non-senescent (CM-NS) cells for the indicated times, and expression of cIAP2 assessed by western blotting. ( G ) Naive GBM cells were treated with BMS-345541 or DMSO as control for 2 h before exposure to CM-SEN ( n = 3 biological replicates comprising 3 technical replicates each), and mean relative expression of BIRC3 + /− SD was assessed by qRT-PCR (a two-tailed Student’s t test was performed; A172 P = 0.000627, 0.000622; U118 P = 0.000001, 0.000001; U87 P = 0.000593, 0.000936, respectively) or ( H ) western blotting for cIAP2. ( I ) GBM cells were exposed to conditioned media from senescent or non-senescent cells ( n = 3) and radiation sensitivity measured by the colony survival assay. The mean percentage of surviving colonies +/− SD ( y axis) is plotted against the corresponding radiation dose ( x axis). .

Article Snippet: BMS-345541 , MedChemExpress , HY-10519.

Techniques: Expressing, Irradiation, Quantitative RT-PCR, Two Tailed Test, Western Blot, Control, Clonogenic Cell Survival Assay