Journal: Cell Proliferation

Article Title: Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off‐target effect of radiotherapy

doi: 10.1111/cpr.13606

Figure Lengend Snippet: Irradiated HMC3 conditioned media (CM) preserve radiation‐naïve U‐87 MG and U‐251 MG cell lines viability. (A) Experimental workflow and CM treatment. (B–I) Cytofluorimetric analysis of viability evaluated with Annexin V/propidium iodide (PI) assay on U‐87 MG treated with U‐87 MG CM (B,C), U‐251 MG treated with U‐251 MG CM (D,E), U‐87 MG treated with HMC3 CM (F,G) and U‐251 MG treated with HMC3 CM (H,I). Data are shown as standard box and whiskers and viability is expressed as the percentage of gated cells, n = 4 independent replicates for each experimental condition. * p ‐value <0.05; *** p ‐value <0.001; **** p ‐value <0.0001. GBM, glioblastoma; Mφ, microglia; IR, irradiated.

Article Snippet: HMC3, were purchased from European Collection of Authenticated Cell Cultures (ECACC, Public Health England, Porton Down Salisbury, UK) and cultured with HMC3 growth medium [Minimum Essential Medium (MEM) supplemented with 10% FBS, Penicillin–Streptomycin 100 IU/mL and L‐glutamine 2 mmol/L].

Techniques: Irradiation

Journal: Cell Proliferation

Article Title: Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off‐target effect of radiotherapy

doi: 10.1111/cpr.13606

Figure Lengend Snippet: Irradiated HMC3 conditioned media (CM) stimulate naïve U‐87 MG and U‐251 MG clone formation. (A,B) Surviving fraction and representative pictures of U‐87 MG treated with 0 Gy and 15 Gy U‐87 MG CM (A) or 0 Gy and 15 Gy HMC3 CM (B). (C,D) Surviving fraction and representative pictures of U‐251 MG treated with 0 Gy and 15 Gy U‐251 MG CM (C) or 0 Gy and 15 Gy HMC3 CM (D). Data are expressed as scattered dot‐plot and mean ± SEM of n ≥ 3 independent experiments. *** p ‐value <0.001 and **** p ‐value <0.0001.

Article Snippet: HMC3, were purchased from European Collection of Authenticated Cell Cultures (ECACC, Public Health England, Porton Down Salisbury, UK) and cultured with HMC3 growth medium [Minimum Essential Medium (MEM) supplemented with 10% FBS, Penicillin–Streptomycin 100 IU/mL and L‐glutamine 2 mmol/L].

Techniques: Irradiation

Journal: Cell Proliferation

Article Title: Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off‐target effect of radiotherapy

doi: 10.1111/cpr.13606

Figure Lengend Snippet: Irradiated HMC3 conditioned media (CM) do not influence mitochondrial mass, fragmentation and integrity in naïve U‐87 MG and U‐251 MG cells. (A,B) Representative pictures of immunofluorescence staining with Mitotracker of U‐87 MG treated with 0 or 15 Gy HMC3 CM (A) and high‐content analysis of Mitotracker mean fluorescence intensity (MFI), mitochondrial fragmentation and percentage of mitochondrial integrity (B). (C,D) Representative pictures of immunofluorescence staining with Mitotracker of U‐251 MG treated with 0 or 15 Gy HMC3 CM (C) and high‐content analysis of Mitotracker MFI, mitochondrial fragmentation and percentage of mitochondrial integrity (D). Data are shown as bar plot and expressed as mean ± SEM of n = 4 independent replicates.

Article Snippet: HMC3, were purchased from European Collection of Authenticated Cell Cultures (ECACC, Public Health England, Porton Down Salisbury, UK) and cultured with HMC3 growth medium [Minimum Essential Medium (MEM) supplemented with 10% FBS, Penicillin–Streptomycin 100 IU/mL and L‐glutamine 2 mmol/L].

Techniques: Irradiation, Immunofluorescence, Staining, High Content Screening, Fluorescence

Journal: Cell Proliferation

Article Title: Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off‐target effect of radiotherapy

doi: 10.1111/cpr.13606

Figure Lengend Snippet: Radiation increases mitochondrial reactive oxygen species and irradiated HMC3 conditioned media (CM) preserve mitochondrial oxidative state of U‐87 MG and U‐251 MG cells. (A) qRT‐PCR analysis of mRNA expression levels of DNM1L, FIS1, MNF1, OPA1, MNF2, CYTB, ND4, TFAM, and ATP5F1A in mock‐IR U‐87 MG, 15 Gy U‐87 MG and mock‐IR treated with 15 Gy U‐87 MG CM or 15 Gy HMC3 CM; data are shown as Log 2 FC over mock‐IR; (B) qRT‐PCR analysis of mRNA expression levels of DNM1L, FIS1, MNF1, OPA1, MNF2, CYTB, ND4, TFAM and ATP5F1A in mock‐IR U‐251 MG, 15 Gy U‐251 MG and mock‐IR treated with 15 Gy U‐251 MG CM or 15 Gy HMC3 CM; data are shown as Log 2 FC over mock‐IR; (C–E) Cytofluorimetric analysis of MitoSOX positive cells in 0 or 15 Gy U‐87 MG (C), 0 or 15 Gy U‐251 MG (D) and 0 or 15 Gy HMC3 (E). (F,G) Cytofluorimetric analysis of MitoSOX positive cells in U‐87 MG cultured with 0 or 15 Gy HMC3 CM (F) and in U‐251 MG cultured with 0 or 15 Gy HMC3 CM (G). Data in (C–G) are shown via standard box and whiskers and are expressed as percentage of MitoSOX positive cells of n = 4 replicates for each experimental condition. * p ‐value <0.05; **** p ‐value <0.0001.

Article Snippet: HMC3, were purchased from European Collection of Authenticated Cell Cultures (ECACC, Public Health England, Porton Down Salisbury, UK) and cultured with HMC3 growth medium [Minimum Essential Medium (MEM) supplemented with 10% FBS, Penicillin–Streptomycin 100 IU/mL and L‐glutamine 2 mmol/L].

Techniques: Irradiation, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Cell Proliferation

Article Title: Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off‐target effect of radiotherapy

doi: 10.1111/cpr.13606

Figure Lengend Snippet: Metformin administration reverses irradiated HMC3 conditioned media (CM)‐induced effects. (A,B) Surviving fraction and representative pictures of U‐87 MG (A) and U‐251 MG (B) cell lines ± metformin, cultured with 15 Gy GBM CM. (C,D) Surviving fraction and representative pictures of U‐87 MG (C) and U‐251 MG (D) cell lines ± metformin, cultured with standard growth medium. (E,F) Surviving fraction and representative pictures of U‐87 MG (E) and U‐251 MG (F) cell lines ± metformin, cultured with 15 Gy HMC3 CM. Data are expressed as scattered dot‐plot and mean ± SEM of n ≥ 4 independent experiments. * p ‐value <0.05 and ** p ‐value <0.01.

Article Snippet: HMC3, were purchased from European Collection of Authenticated Cell Cultures (ECACC, Public Health England, Porton Down Salisbury, UK) and cultured with HMC3 growth medium [Minimum Essential Medium (MEM) supplemented with 10% FBS, Penicillin–Streptomycin 100 IU/mL and L‐glutamine 2 mmol/L].

Techniques: Irradiation, Cell Culture

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a: Schematic of the screen. Three sgRNAs were designed to target each of 74 candidate regulatory regions (CREs) from our V2G mapping effort. We created a CRISPRi helper line expressing dCas9-KRAB in HMC3 cells and transduced it with a lentiviral pool of the sgRNAs at low MOI so that most cells would only receive a single perturbation. After selection and expansion, we performed scRNA-seq with 10x Genomics. Upon successful targeting of a CRE using CRISPRi, we anticipate RNA expression knockdown of the target gene. b, c, d: QQ plots from SCEPTRE differential expression analysis showing b ) good calibration, c) significant signal, and d ) volcano plot of the hits. These plots refer to the V2G “union” analysis. e , f , g : a candidate region containing three AD-associated SNPs (rs7080009, rs1870138, and rs1870137) regulates TSPAN14 expression in microglial but not in neuronal cells. CRISPRi ( e ) and CRISPRa ( f ) were performed in HMC3 cells stably expressing dCas9-KRAB and dCas9-VPR, respectively, via lentiviral delivery of three gRNAs targeting the region (T1, T2, and T3) and three non-targeting control guides (N1, N2, and N3). TSPAN14 expression was assessed by qPCR (N=3; bar plots show mean TSPAN14 expression with SEM error bars normalized to their respective control line with no guide. Significance by one-way ANOVA followed by pairwise t-test. ** P <5×10 −5 ; * P <0.05. A similar CRISPRi experiment was performed in the neuronal ReNcell VM line, showing no effect on TSPAN14 expression ( c ). P: a guide targeting TSPAN14 promoter was used as a positive control.

Article Snippet: 2ml of cell culture media were collected from each 6 well on the second day after transfection, filtered using 0.45um syringe filters (Fisher Scientific, cat# 13100107) and applied to a 60% confluent 10cm plate with HMC3 cells in the presence of 8μg/ml polybrene (Fisher Scientific, cat# TR1003G) as following: 2ml of lentiCRISPRv2puro-right guide and lentiCRISPRv2mCherry-left guide virus-containing media were mixed with 8 ml of cell culture media for HMC3 cells (EMEM, ATCC, cat# 30-2003, with 10% FBS Fisher Scientific, cat# MT35010CV) and 8μl of 10mg/ml MilliporeSigma polybrene transfection reagent (Fisher Scientific, cat# TR1003G).

Techniques: Expressing, Selection, RNA Expression, Knockdown, Quantitative Proteomics, Stable Transfection, Control, Positive Control

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a: genomic location of the three AD SNPs (red) showing the chromatin loop with the TSPAN14 promoter. b, c : in luciferase assays, a 1.6 kb intronic region containing the three AD SNPs increases reporter luminescence in microglial cells ( b ; HMC3 cells; two-sample t-test P =8×10 −4 ), but not in neuronal cells ( c ; SH-SY5Y cells). minP: minimal promoter only (no enhancer region). NNN: enhancer region with 3 SNPs with non-risk alleles. d : constructs containing AD risk alleles significantly increase enhancer activity compared to TSPAN14 promoter alone (* P <0.05, ** P <0.005), while a construct with all non-risk allele does not. Cell line: HMC3; DNN: rs7080009 risk allele only; NDN: rs1870138 risk allele only; NND: rs1870137 risk allele only; DDD: 3 SNPs with risk alleles; Prom: TSPAN14 promoter only (no enhancer region). Bar plots show mean reporter activity with SEM error bars normalized to promoter only control. Statistical analysis by one-way ANOVA ( P =1.7×10 −3 ) followed by pairwise t-test with BH correction, N=3. e, f, g: rs7080009, rs1870138, and rs1870137 AD-risk alleles affect binding of specific transcription factors in microglial cells. TF binding analysis was performed with motifbreakR using the “default” scoring method. In each panel, sequence logo diagrams for TF binding site motifs matching each SNP locus are plotted with respect to the reference genome (top, non-risk allele; bottom, risk allele). Motifs on top have stronger binding to the non-risk allele and motifs on bottom to the risk allele; both are ordered by the magnitude of the difference in binding affinities. The name of the TF is adjacent to each motif. Motifs depicted have expression > 10 TPM in at least two of the three cell lines analyzed (HMC3, iMg, monocytes).

Article Snippet: 2ml of cell culture media were collected from each 6 well on the second day after transfection, filtered using 0.45um syringe filters (Fisher Scientific, cat# 13100107) and applied to a 60% confluent 10cm plate with HMC3 cells in the presence of 8μg/ml polybrene (Fisher Scientific, cat# TR1003G) as following: 2ml of lentiCRISPRv2puro-right guide and lentiCRISPRv2mCherry-left guide virus-containing media were mixed with 8 ml of cell culture media for HMC3 cells (EMEM, ATCC, cat# 30-2003, with 10% FBS Fisher Scientific, cat# MT35010CV) and 8μl of 10mg/ml MilliporeSigma polybrene transfection reagent (Fisher Scientific, cat# TR1003G).

Techniques: Luciferase, Construct, Activity Assay, Control, Binding Assay, Sequencing, Expressing

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a: via CRISPR-Cas9 editing, we generated two clonal HMC3 lines harboring ∼780 bp homozygous genomic deletions spanning the three AD SNPs. b: DNA agarose gel shows sizes of the PCR amplicons for clones 1 and 2 and a control clone with no deletion. c: TSPAN14 expression levels by qPCR in clones 1 and 2 (blue) and control lines (grey). TSPAN14 expression was significantly decreased (60-80%) in both deletion clones (two-sample t-test P =2×10 −3 ). NTC1, NTC2, NTC3: control lines, each with a different non-targeting guide. N=3. Bar plots show mean TSPAN14 expression levels with SEM error bars normalized to the average of the control lines. d: Volcano plot showing differentially expressed genes in the same two KO lines vs three control lines expressing non-targeting guides. The dashed lines show thresholds for Fold Change (1.5) and FDR (0.05). e: PANTHER enrichment analysis using the GO-Slim Biological Process annotation set. Top (red): significantly upregulated pathways. Bottom (blue): significantly downregulated pathways. Correction: FDR<0.05. For each significantly enriched term, only the top term of the GO hierarchy (i.e., the most specific) is shown.

Article Snippet: 2ml of cell culture media were collected from each 6 well on the second day after transfection, filtered using 0.45um syringe filters (Fisher Scientific, cat# 13100107) and applied to a 60% confluent 10cm plate with HMC3 cells in the presence of 8μg/ml polybrene (Fisher Scientific, cat# TR1003G) as following: 2ml of lentiCRISPRv2puro-right guide and lentiCRISPRv2mCherry-left guide virus-containing media were mixed with 8 ml of cell culture media for HMC3 cells (EMEM, ATCC, cat# 30-2003, with 10% FBS Fisher Scientific, cat# MT35010CV) and 8μl of 10mg/ml MilliporeSigma polybrene transfection reagent (Fisher Scientific, cat# TR1003G).

Techniques: CRISPR, Generated, Agarose Gel Electrophoresis, Clone Assay, Control, Expressing

Journal: bioRxiv

Article Title: Integration of Alzheimer’s GWAS, 3D genomics, and single-cell CRISPRi non-coding screen implicates causal variants in a microglial enhancer regulating TSPAN14

doi: 10.1101/2025.04.01.646442

Figure Lengend Snippet: a, b: Secreted IL-6 and IL-8 protein levels are strongly reduced in TSPAN14 enhancer KO microglial cells with deletions encompassing AD-risk variants rs7080009, rs1870138, and rs1870137. ELISA was performed on cell culture media of two HMC3 clonal lines (clone 1 and 2, blue). IL-6 or IL-8 protein levels are normalized to the amounts of total protein in each well. Controls are lines harboring a non-targeting guide and/or an empty vector (grey). Bar plots show means with SEM error bars; N=3. *** P <5×10 −3 ; two-sample t-test. c : The same KO lines with TSPAN14 enhancer deletions (blue) showed a significant reduction (∼25-30%) in intensity of phalloidin (F-actin) staining as compared to three control lines (grey). Single cells were segmented, quantified, and the fluorescence intensities of all cells in each well were averaged. Three wells were measured per condition, with 200-500 cells quantified per well. Bar plots show average fluorescence intensity with SEM error bars. *** P <5×10 −3 ; two-sample t-test. d: Representative images of NTC1 and clone 1 are shown, with notably less F-actin staining observed for clone 1, which harbors the TSPAN14 enhancer deletion.

Article Snippet: 2ml of cell culture media were collected from each 6 well on the second day after transfection, filtered using 0.45um syringe filters (Fisher Scientific, cat# 13100107) and applied to a 60% confluent 10cm plate with HMC3 cells in the presence of 8μg/ml polybrene (Fisher Scientific, cat# TR1003G) as following: 2ml of lentiCRISPRv2puro-right guide and lentiCRISPRv2mCherry-left guide virus-containing media were mixed with 8 ml of cell culture media for HMC3 cells (EMEM, ATCC, cat# 30-2003, with 10% FBS Fisher Scientific, cat# MT35010CV) and 8μl of 10mg/ml MilliporeSigma polybrene transfection reagent (Fisher Scientific, cat# TR1003G).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Plasmid Preparation, Staining, Control, Fluorescence