Journal: Cell Death Discovery

Article Title: Novel mutations in the RECQL4 gene affect its helicase functions, interactions with the BLM helicase and chemotherapeutics-induced cell death

doi: 10.1038/s41420-025-02834-w

Figure Lengend Snippet: A Representative immunoblots show GFP-tagged WT RECQL4 (RQ4wt), RECQL4 P532S (RQ4m1) and RECQL4 R766Q (RQ4m2) proteins overexpressed in human RQ4 KO LN18 and LN229 glioma cells detected by Western blotting. The presence of GFP resulted in a high molecular weight of the RECQL4 proteins. The levels of BLM helicase and a-tubulin are shown. B Cell viability was determined with PRESTO Blue 48 h after transfection of RECQL4 KO cells with the tested RECQL4 constructs. The results are shown as mean ± SD, n = 4, in triplicates. C , D Immunoprecipitation experiment showing distinct binding RQ4 variants to its partner BLM in LN18 and LN229 glioma cells ( C ) and its quantification ( D ). RECQL4 deficient cells were transfected with the tested RECQL4 constructs and 48 h after transfection cells were lysed, total protein extracts isolated and subjected to immunoprecipitation (IP) with anti-GFP antibody. IP extracts were resolved by SDS-PAGE and subjected to Western blotting. Immunoblots were quantified by densitometry, results are presented as mean ± SD, n = 3. Statistical analysis was performed on log raw data using one-way ANOVA with Tukey’s post-hoc test (# p < 0.001). Uncropped Western blots are presented in the Supplemental material.

Article Snippet: LN18 and LN229 glioma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM medium (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific; Waltham, MA, USA).

Techniques: Western Blot, High Molecular Weight, Transfection, Construct, Immunoprecipitation, Binding Assay, Isolation, SDS Page

Journal: Cell Death Discovery

Article Title: Novel mutations in the RECQL4 gene affect its helicase functions, interactions with the BLM helicase and chemotherapeutics-induced cell death

doi: 10.1038/s41420-025-02834-w

Figure Lengend Snippet: A, B Immunofluorescent staining showing localization of BLM, different GFP-RECQL4 variants and γH2AX in RECQL4 KO LN18 (A) and LN229 (B) glioma cells under basal conditions and after exposure to UV-C. Nuclei were visualized by DAPI staining and γH2AX foci accumulate at the sites of double stand DNA breaks. White arrow indicate BLM sequestered in the cytoplasm in UV-C treated cells transfected with GFP-tagged RECQL4 P532S (RQ4m1) and RECQL4 R766Q (RQ4m2) proteins. Scale bar 50 µM. Images are representative of 3 independent experiments.

Article Snippet: LN18 and LN229 glioma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM medium (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific; Waltham, MA, USA).

Techniques: Staining, Transfection

Journal: Cell Death Discovery

Article Title: Novel mutations in the RECQL4 gene affect its helicase functions, interactions with the BLM helicase and chemotherapeutics-induced cell death

doi: 10.1038/s41420-025-02834-w

Figure Lengend Snippet: A , B Immunofluorescent staining showing expression of BLM, and γH2AX in RECQL4 KO LN18 (A) and LN229 ( B ) glioma cells overexpressing GFP-RECQL4 variants under basal conditions and after exposure to 500 µM TMZ for 48 h. TMZ induces γH2AX accumulation in KO cells transfected with GFP-tagged wild type RECQL4 (RQ4wt) but not in cells expressing RECQL4 P532S (RQ4m1) and RECQL4 R766Q (RQ4m2) proteins. TMZ induces a prominent γH2AX accumulation in RECQL4 KO LN18 cells and moderate γH2AX accumulation in RECQL4 KO LN229 cells. Scale bar 50 µM. Images are representative of 3 independent experiments. C , D Quantification of BLM and γH2AX in RECQL4 KO LN18 ( C ) and LN229 (D ) glioma cells overexpressing GFP-RECQL4 variants. Immunofluorescence signal intensity was measured. C BLM signal intensity is significantly increased in TMZ-treated RECQL4 KO LN18 cells expressing RECQL4 R766Q (RQ4m2) proteins. TMZ-induced γH2AX accumulation in LN18 RECQL4 KO cells is counteracted by transfection with RQ4wt but not with RECQL4 P532S (RQ4m1) and RECQL4 R766Q (RQ4m2). D TMZ does not induces similar changes in LN229 that show γH2AX accumulation after TMZ treatment. Results are presented as mean ± SD, n = 3. WT –parental cells. Statistical analysis was performed on raw data using two-way ANOVA with Tukey’s post-hoc test (within the groups) or Sidak’s post-hoc test (between DMSO and TMZ groups) (*p < 0.05, & p < 0.01). E , F Cell viability of RECQL4 KO LN18 ( E ) and LN229 ( F ) glioma cells overexpressing GFP-RECQL4 variants and treated with TMZ. Cell viability was determined with Presto Blue. Results are presented as mean ± SD, n = 3. WT –parental cells. Statistical analysis was performed on raw data using one-way ANOVA with Dunnett’s post-hoc test (*p < 0.05).

Article Snippet: LN18 and LN229 glioma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM medium (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific; Waltham, MA, USA).

Techniques: Staining, Expressing, Transfection, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: EF24 targets METTL3 to reprogram m6A methylation and induce ferroptosis: an epitranscriptomic mechanism with therapeutical potential for glioma

doi: 10.1186/s12964-025-02583-4

Figure Lengend Snippet: EF24 inhibits glioma cell proliferation. A CCK-8 assay showing the inhibitory effect of EF24 on glioma cell viability and the IC50 values for LN229 and A172 cells. B Plate colony formation assay assessing the effect of EF24 on glioma cell proliferation. C Immunofluorescence analysis of the effect of EF24 on glioma cell proliferation (magnification ×200, scale bar: 50 μm). Note: * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the control group

Article Snippet: Human glioma cell lines LN229 (RRID: CVCL-0393) and A172 (RRID: CVCL-0131) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA), cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin antibiotic cocktail (10,000 U/mL penicillin, 10 mg/mL streptomycin).

Techniques: CCK-8 Assay, Colony Assay, Immunofluorescence, Control

Journal: Cell Communication and Signaling : CCS

Article Title: EF24 targets METTL3 to reprogram m6A methylation and induce ferroptosis: an epitranscriptomic mechanism with therapeutical potential for glioma

doi: 10.1186/s12964-025-02583-4

Figure Lengend Snippet: METTL3 inhibits the ferroptosis in glioma cells. A Western blotting analysis of NRF2 and GPX4 in LN229 and A172 cells in the negative control group and the METTL3 knockdown group. B Correlation analysis of METTL3 expression with NRF2 and GPX4 mRNA levels in GBM tissue specimens. C Detection of intracellular malondialdehyde (MDA) levels after METTL3 knockdown. D Intracellular glutathione (GSH) levels following METTL3 knockdown. E TEM images showing mitochondrial morphological changes in METTL3-knockdown cells (magnification: ×7000, scale bar = 2.0 μm; magnification: ×20000, scale bar = 50 μm). F Detection of glioma proliferation following METTL3 knockdown by colony formation assay. Note: * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control group

Article Snippet: Human glioma cell lines LN229 (RRID: CVCL-0393) and A172 (RRID: CVCL-0131) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA), cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin antibiotic cocktail (10,000 U/mL penicillin, 10 mg/mL streptomycin).

Techniques: Western Blot, Negative Control, Knockdown, Expressing, Colony Assay, Control

Journal: Cell Communication and Signaling : CCS

Article Title: EF24 targets METTL3 to reprogram m6A methylation and induce ferroptosis: an epitranscriptomic mechanism with therapeutical potential for glioma

doi: 10.1186/s12964-025-02583-4

Figure Lengend Snippet: METTL3 recruits YTHDF1 to regulate NRF2 mRNA stability. A Actinomycin assay showing the effect of YTHDF1 on NRF2 stability. B qRT-PCR showing the effect of knockdown of YTHDF1 on NRF2 expression. C Correlation analysis of YTHDF1 and NRF2 expression in GBM tissue specimens. D Western blotting showing the effect of knockdown of YTHDF1 on NRF2 expression. E RIP-qPCR revealing the binding of YTHDF1 with NRF2 in stable METTL3 knockdown and negative control LN229 and A172 cells. Note: * P < 0.05, ** P < 0.01, *** P < 0.001 compared to control

Article Snippet: Human glioma cell lines LN229 (RRID: CVCL-0393) and A172 (RRID: CVCL-0131) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA), cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin antibiotic cocktail (10,000 U/mL penicillin, 10 mg/mL streptomycin).

Techniques: Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Binding Assay, Negative Control, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone demethylase KDM9 activates the CCND1/AKT pathway to promote the malignant progression of gliomas through interaction with BRD2

doi: 10.1007/s00018-025-05900-9

Figure Lengend Snippet: KDM9 promotes malignant progression and reduces TMZ sensitivity in glioma. After using LN229 and SW1088 cells with knocked down KDM9 (LN229-shNC, LN229-shKDM9#1, LN229-shKDM9#2, SW1088-shNC, SW1088-shKDM9#1, SW1088-shKDM9#2) or the transfection of pcDNA3.1-KDM9 plasmid (KDM9-OE) in U251 cells for 48 h, ( A ) cell proliferation was measured via the CCK-8 assay. ( B ) Cell invasion was evaluated via the transwell assay. ( C ) The sphere-formation assay was conducted to assess the stem-like characteristics of glioma cells. ( D ) A Western blot experiment was performed to examine the expression of the stemness markers Nestin and CD133. ( E ) TMZ sensitivity was assessed via the colony formation assay after treatment with TMZ (100 μM) in glioma cells. ( F – H ) LN229-shKDM9 cells (1 × 10 7 ) were injected subcutaneously into nude mice to establish xenograft models ( N = 9). Tumor size ( F ), tumor volume ( G ), and tumor weight ( H ) were measured. ( I ) H&E and IHC staining were performed to detect the protein expression of KDM9 and Ki67 in the tumor tissues of nude mice. NC: negative control of shRNA, Vec: blank plasmid. Data are shown as the mean ± SD. ** P < 0.01 and *** P < 0.001

Article Snippet: The normal human brain glial cells HEB; the human glioma cells LN229 (ATCC: CRL-2611), SW1088 (ATCC: HTB-12), and T98G (ATCC: CRL-1690), U118 (ATCC: HTB-15), U251, and human embryonic kidney cells 293 T (ATCC: CRL-11268) were obtained from the Cell Center of Central South University.

Techniques: Transfection, Plasmid Preparation, CCK-8 Assay, Transwell Assay, Tube Formation Assay, Western Blot, Expressing, Colony Assay, Injection, Immunohistochemistry, Negative Control, shRNA

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone demethylase KDM9 activates the CCND1/AKT pathway to promote the malignant progression of gliomas through interaction with BRD2

doi: 10.1007/s00018-025-05900-9

Figure Lengend Snippet: H4K20me2 demethylation was mediated by KDM9 in the CCND1 promotor region to promote its transcription. RNA-Seq was performed on LN229 cells with knocked down KDM9, ( A ) Volcano plots of differential genes are shown, along with ( B ) bubble plots of enriched signaling pathways for differentially expressed genes. ( C ) ChIP-Seq was conducted using the H4K20me2 antibody in LN229 cells, and the distribution of reads around the transcription start site (TSS) was displayed. ( D ) The intersection between genes enriched in the PI3K/AKT pathway from RNA-Seq and genes obtained from ChIP-Seq is shown by a Venn diagram. ( E ) The intersecting genes are shown on a clustering heatmap. ( F , G ) qPCR was performed to detect mRNA expression of BCL2L11, BCL2, CCND1, and CREB3L2 in glioma cells after the knockdown or overexpression of KDM9. ( H ) Western blot experiment was performed to detect the protein expression of CCND1 in glioma cells with the knockdown or overexpression of KDM9. ( I ) Dual-luciferase reporter assay was performed in 293 T cells with KDM9 knockdown to detect CCND1 transcription activity. ( J ) Schematic diagram of the ChIP-qPCR primer (designed around the transcription start site of CCND1 from −2000 to + 300 bp). ( K ) ChIP-qPCR was used to measure the enrichment level of H4K20me2 at the CCND1 P4 promotor region (IgG was used as a negative control). NC: negative control for shRNA; Vec: blank plasmid. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The normal human brain glial cells HEB; the human glioma cells LN229 (ATCC: CRL-2611), SW1088 (ATCC: HTB-12), and T98G (ATCC: CRL-1690), U118 (ATCC: HTB-15), U251, and human embryonic kidney cells 293 T (ATCC: CRL-11268) were obtained from the Cell Center of Central South University.

Techniques: RNA Sequencing, Protein-Protein interactions, ChIP-sequencing, Expressing, Knockdown, Over Expression, Western Blot, Luciferase, Reporter Assay, Activity Assay, ChIP-qPCR, Negative Control, shRNA, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone demethylase KDM9 activates the CCND1/AKT pathway to promote the malignant progression of gliomas through interaction with BRD2

doi: 10.1007/s00018-025-05900-9

Figure Lengend Snippet: KDM9 regulates CCND1 transcription through interaction with BRD2. ( A ) Protein silver staining of IP via KDM9 antibody enrichment in LN229 cells. ( B ) Transcription factors in the protein mass spectrometry data are shown. Exogenous and endogenous IP experiments were performed to analyze the interaction between KDM9 and BRD4 ( C ) or BRD2 ( D ). ( E ) The IF assay was used to detect the expression and co-localization of KDM9 and BRD2 in LN229 cells. ( F ) After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, ChIP-qPCR was performed to detect the enrichment of H4K20me2 at the CCND1 P4 promoter region (with IgG as negative control). ( G ) After the transfection of wild-type or mutant KDM9 plasmids into U251 cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity (Left). After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, luciferase reporter assays were performed to detect CCND1 transcription activity (Right). ( H ) After the transfection of shBRD2 plasmids into U251-KDM9-WT cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity. ( I ) After the transfection of pcDNA3.1-BRD2-His into shKDM9 glioma cells for 48 h, a Western blot experiment was conducted to detect the protein expression of KDM9, BRD2, CCND1, AKT/P-AKT, mTOR/P-mTOR, and CD133. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The normal human brain glial cells HEB; the human glioma cells LN229 (ATCC: CRL-2611), SW1088 (ATCC: HTB-12), and T98G (ATCC: CRL-1690), U118 (ATCC: HTB-15), U251, and human embryonic kidney cells 293 T (ATCC: CRL-11268) were obtained from the Cell Center of Central South University.

Techniques: Silver Staining, Mass Spectrometry, Expressing, Transfection, ChIP-qPCR, Negative Control, Mutagenesis, Luciferase, Activity Assay, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone demethylase KDM9 activates the CCND1/AKT pathway to promote the malignant progression of gliomas through interaction with BRD2

doi: 10.1007/s00018-025-05900-9

Figure Lengend Snippet: KDM9 promotes glioma growth and reduces TMZ sensitivity in vivo. ( A - C ) LN229-shKDM9 or LN229-shNC cells (1 × 10 7 ) were injected subcutaneously into nude mice to establish xenograft models ( N = 6); after the tumor grew to 60-80 mm 3 , TMZ (40 mg/kg) was injected intraperitoneally for 5 consecutive days, and the tumor size ( A ), tumor volume ( B ) and tumor weight ( C ) were measured. ( D ) IHC staining was performed to detect the protein expression of KDM9, H4K20me2, CCND1, P-AKT, and CD133 in nude mouse tumor tissues. ( E ) The bioluminescence images of intracranial injection of LN229-luc-shKDM9 or LN229-luc-shNC cells or/and TMZ treatment in mice ( N = 6). The scale bar for bioluminescence intensity is shown on the right. ( F ) The quantification of the bioluminescence intensity of tumors. ( G ) Representative images of mouse brains stained with H&E at the termination of the experiment. ( H ) Kaplan–Meier survival curves of mice. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The normal human brain glial cells HEB; the human glioma cells LN229 (ATCC: CRL-2611), SW1088 (ATCC: HTB-12), and T98G (ATCC: CRL-1690), U118 (ATCC: HTB-15), U251, and human embryonic kidney cells 293 T (ATCC: CRL-11268) were obtained from the Cell Center of Central South University.

Techniques: In Vivo, Injection, Immunohistochemistry, Expressing, Staining