anti recql4 ab  (Novus Biologicals)

Journal: Cancers

Article Title: Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

doi: 10.3390/cancers12102919

Figure Lengend Snippet: RECQL4 expression is upregulated in human malignant gliomas. ( A ) RECQL4 expression in normal brain (NB), WHO grade II and grade III gliomas and glioblastomas (GBM, WHO grade IV) in TCGA datasets. Presented values are log 2 of FPKM values. Statistical significance was determined by Welch’s analysis of variance (ANOVA) between GII, GIII and GIV groups. ( B ) Quantitative analysis of RECQL4 mRNA levels in NB ( n = 9), and gliomas of different grades: GI ( n = 25), GII/III ( n = 29) and GBM ( n = 50). The RECQL4 expression was normalized to GAPDH ; results represent means ± SEM; statistical significance was determined by one-way ANOVA, followed by Dunnett’s post hoc test. p -Values were considered as significant when * p < 0.05. ( C ) Kaplan–Meier overall survival analysis of LGG and GBM patients from TCGA. Log-rank test was calculated between RECQL4 LOW and HIGH expression groups (* p < 0.05). ( D ) Representative immunostaining showing expression of RECQL4 protein in the glioma tissue microarray including astrocytomas ( n = 132), glioblastomas ( n = 31), oligoastrocytomas ( n = 7), oligodendrogliomas ( n = 9), ependymomas ( n = 11), ganglioglioma ( n = 1) and gliosarcoma ( n = 1), plus tumour adjacent and normal brain (NB) tissues ( n = 8). RECQL4 expression appeared as high nuclear and low cytoplasmic signal (upper panel) and low nuclear and high cytoplasmic signal (bottom panel). ( E ) Quantification of RECQL4 immunoreactivity. Statistical significance was determined by chi-squared test. p -values of NB p = 1.0, GI p = 0.019, GII p = 1.1 × 10 −5 , GIII p = 6.3 × 10 −5 , GIV p = 0.011. ( F ) Quantitative analysis of RECQL4 mRNA levels in established glioma cell lines, patient-derived primary cultures, and normal human astrocytes (NHA). The expression was normalized to GAPDH ; results represent means ± SEM of 3 cell passages ( n =3). Statistical significance was determined by one-way ANOVA. p -Values were considered as significant when * p < 0.05, ** p < 0.01, *** p < 0.001. ( G ) Representative immunoblot shows RECQL4 expression in established glioma cell lines and GBM primary cultures in comparison to NHA. β-Actin was used as a loading control.

Article Snippet: Cells were seeded on coverslips (3 × 10 4 cells/well) for 30 h. The medium was changed to PBS (with Ca 2+ and Mg 2+ ) to induce starvation conditions and the cells were cultured for 18 h. To visualize mitochondrial network, cells were stained with 250 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in dark at 37 °C for 20 min, washed 3 times with a complete medium and 3 times with a PBS, fixed with 4% PFA/PBS pH 7.4 for 15 min and permeabilised with 100% methanol for 10 min. Unspecific signals were blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in 0.3% Triton X-100/PBS for 40 min at RT, followed by incubation with anti-RECQL4 Ab (1:200, Novus Biological) in 3% NDS, 1% BSA in 0.3% Triton X-100/PBS at +4 °C overnight.

Techniques: Expressing, Immunostaining, Microarray, Derivative Assay, Western Blot, Comparison, Control

Journal: Cancers

Article Title: Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

doi: 10.3390/cancers12102919

Figure Lengend Snippet: RECQL4 depletion affects cell viability and proliferation of certain glioblastoma cells. ( A ) Representative immunoblots showing effective knockdown of RECQL4 (48 h post-transfection) with two different siRNAs (siRQ) in LN18 (upper panel) and U87-MG (bottom panel) cells. β-Actin was used as a loading control. Viability ( B ) and proliferation ( C ) of LN18 and U87-MG 48 h post-transfection was assessed by MTT metabolism and BrdU incorporation tests, respectively. The results were normalized to cells transfected with the non-targeting control siRNA (siCTRL) and represent the mean ± SEM ( n = 4). Statistical significance was determined with by two-tailed t-test; statistically significant when * p < 0.05. ( D , E ) Representative immunoblots show knockout of RECQL4 by CRISPR/Cas9 in two independent clones (RECQL4 KO #1 and KO #2) of LN18 and LN229 cells. Viability ( G , H ) and proliferation ( F–I ) of LN18 or LN229 cells were assessed 48 h and 72 h after seeding as described above. The results were normalized to wild type (WT) cells and represent the mean ± SEM of four independent experiments. Statistical significance was determined by one sample t-test; significant when * p < 0.05, ** p < 0.01.

Article Snippet: Cells were seeded on coverslips (3 × 10 4 cells/well) for 30 h. The medium was changed to PBS (with Ca 2+ and Mg 2+ ) to induce starvation conditions and the cells were cultured for 18 h. To visualize mitochondrial network, cells were stained with 250 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in dark at 37 °C for 20 min, washed 3 times with a complete medium and 3 times with a PBS, fixed with 4% PFA/PBS pH 7.4 for 15 min and permeabilised with 100% methanol for 10 min. Unspecific signals were blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in 0.3% Triton X-100/PBS for 40 min at RT, followed by incubation with anti-RECQL4 Ab (1:200, Novus Biological) in 3% NDS, 1% BSA in 0.3% Triton X-100/PBS at +4 °C overnight.

Techniques: Western Blot, Knockdown, Transfection, Control, BrdU Incorporation Assay, Two Tailed Test, Knock-Out, CRISPR, Clone Assay

Journal: Cancers

Article Title: Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

doi: 10.3390/cancers12102919

Figure Lengend Snippet: RNA-seq reveals gross transcriptomic changes in RECQL4-depleted glioma cells. Total RNA from WT and RECQL4 KO#2 glioma cells were subjected to RNA sequencing. KEGG analysis of DE genes (FDR corrected p < 0.05) shows genes/pathways downregulated ( A ) or upregulated ( B ) in RECQ4 KO cells when compared to WT cells. ( C ) Volcano plot shows down- and upregulated genes (log 2 fold change < 0 and log 2 fold change > 0, respectively, and FDR-correction p < 0.05) in RECQ4 KO cell lines relative to the WT. Genes from selected functional KEGG categories ( q -value < 0.05) as marked by red arrows on panel ( B ) are marked in different colours, as indicated in the legend in bottom left of the panel ( C ). Selected KEGG pathways are also presented on z-score heatmaps: apoptosis ( D ) focal adhesion ( E ) nucleotide excision repair ( F ) mismatch repair ( G ) DNA replication ( H ) and p53 signalling pathway ( I ).

Article Snippet: Cells were seeded on coverslips (3 × 10 4 cells/well) for 30 h. The medium was changed to PBS (with Ca 2+ and Mg 2+ ) to induce starvation conditions and the cells were cultured for 18 h. To visualize mitochondrial network, cells were stained with 250 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in dark at 37 °C for 20 min, washed 3 times with a complete medium and 3 times with a PBS, fixed with 4% PFA/PBS pH 7.4 for 15 min and permeabilised with 100% methanol for 10 min. Unspecific signals were blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in 0.3% Triton X-100/PBS for 40 min at RT, followed by incubation with anti-RECQL4 Ab (1:200, Novus Biological) in 3% NDS, 1% BSA in 0.3% Triton X-100/PBS at +4 °C overnight.

Techniques: RNA Sequencing, Functional Assay

Journal: Cancers

Article Title: Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

doi: 10.3390/cancers12102919

Figure Lengend Snippet: RECQL4 supports self-renewal of glioma stem cells and their resistance to temozolomide. ( A ) Pearson’s correlation between expression of RECQL4 and selected stem cell markers in the TCGA dataset showed significant positive correlation of RECQL4 and MYC, NES, PROM1 and MSI1 genes. ( B ) qPCR analysis of the RECQL4 mRNA level in LN18 spheres and parental adherent cells. The expression was normalized to GAPDH ; the results represent mean ± SEM ( n = 7 different sphere cultures). Statistical significance was determined by two-tailed paired t -test. p -Values were considered as significant when * p < 0.05. ( C ) Representative immunoblot showing RECQL4 expression in LN18 spheres and adherent cells. β-Actin was used as a loading control. ( D) Representative images and quantification of LN18 spheres (at day 7th) after siRNA-mediated RECQL4 knockdown compared to control siRNA transfected spheres. Scale bar represents 200 µm. The results are shown as numbers of spheres (≥100 µm in diameter were counted) and represent means ± SEM ( n = 4). Statistical significance was determined by two-tailed paired t-test. p -Values were considered as significant when * p < 0.05. ( E ) Representative images and quantification of (at day 7th) WT and RECQL4 knockout LN18 spheres. ( F ) Representative immunoblots showing analysis of cell death, proliferation and cell cycle proteins in WT and RECQL4 KO LN18 spheres. ( G ) Quantification of spheres in WT and RECQL4 KO cultures exposed to 500 µM TMZ for 72 h. The results are shown as numbers of spheres and represent the means ± SEM ( n = 3). Statistical significance was determined by RM two-way ANOVA, with followed by Tukey’s HSD post hoc test. P values were considered as significant when * p < 0.05, ** p < 0.01, *** p < 0.001. ( H ) Representative immunoblots show the levels of cell death, proliferation and cell cycle proteins in WT and RECQL4 KO LN18 spheres 72 h after TMZ treatment.

Article Snippet: Cells were seeded on coverslips (3 × 10 4 cells/well) for 30 h. The medium was changed to PBS (with Ca 2+ and Mg 2+ ) to induce starvation conditions and the cells were cultured for 18 h. To visualize mitochondrial network, cells were stained with 250 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in dark at 37 °C for 20 min, washed 3 times with a complete medium and 3 times with a PBS, fixed with 4% PFA/PBS pH 7.4 for 15 min and permeabilised with 100% methanol for 10 min. Unspecific signals were blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in 0.3% Triton X-100/PBS for 40 min at RT, followed by incubation with anti-RECQL4 Ab (1:200, Novus Biological) in 3% NDS, 1% BSA in 0.3% Triton X-100/PBS at +4 °C overnight.

Techniques: Expressing, Two Tailed Test, Western Blot, Control, Knockdown, Transfection, Knock-Out

Journal: Cancers

Article Title: Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

doi: 10.3390/cancers12102919

Figure Lengend Snippet: RECQL4 depletion affects morphology and functions of mitochondrial network in glioblastoma cells. ( A ) Representative immunoblots showing subcellular distribution of RECQL4 in cytosolic (Cyto) and mitochondrial (Mito) fractions of human U87-MG, LN18 and primary WG4, IPIN cultures of glioblastoma cells. Expression of GAPDH and Tomm20 was used as a loading control and markers for cytosolic and mitochondrial fractions, respectively. ( B ) Representative images of mitochondrial morphology and network organisation in LN18 (upper panel) and LN229 (bottom panel) cells analysed by confocal microscopy. WT and RECQL4 KO cells were stained for mitochondria (MitoTracker, red) and nuclei (DAPI, blue). Scale bar represents 5 µm. ( C ) Representative histograms and quantification of mitochondrial transmembrane potential changes in WT and KO LN18 cells. The cells were stained with JC-1 probe and analysed by FACS. CCCP was used as a positive control. The results are shown as red/ green fluorescence intensity ratio (relative FL-2/ FL-1) in RECQL4 KO cells normalized to WT cells, and represent means ± SEM ( n = 3). Statistical significance was determined by RM two-way ANOVA, with followed by Tukey’s HSD post hoc test. P values were considered as significant when * p < 0.05, ** p < 0.01. ( D ) Representative histograms and quantification of mitochondrial transmembrane potential changes in WT and RECQL KO LN229 cells.

Article Snippet: Cells were seeded on coverslips (3 × 10 4 cells/well) for 30 h. The medium was changed to PBS (with Ca 2+ and Mg 2+ ) to induce starvation conditions and the cells were cultured for 18 h. To visualize mitochondrial network, cells were stained with 250 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in dark at 37 °C for 20 min, washed 3 times with a complete medium and 3 times with a PBS, fixed with 4% PFA/PBS pH 7.4 for 15 min and permeabilised with 100% methanol for 10 min. Unspecific signals were blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in 0.3% Triton X-100/PBS for 40 min at RT, followed by incubation with anti-RECQL4 Ab (1:200, Novus Biological) in 3% NDS, 1% BSA in 0.3% Triton X-100/PBS at +4 °C overnight.

Techniques: Western Blot, Expressing, Control, Confocal Microscopy, Staining, Positive Control, Fluorescence

Journal: Cancers

Article Title: Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

doi: 10.3390/cancers12102919

Figure Lengend Snippet: RECQL4 deficiency sensitises LN18 glioblastoma cells to temozolomide or olaparib. WT and RECQL4 KO LN18 cells were exposed to TMZ at concentrations of 250 and 500 µM for 72 h, and cell viability ( A ) and proliferation ( B ) were analysed. The results were normalized to untreated cells (CTRL) and represent means ± SEM ( n = 4). Statistical significance was determined by RM two-way ANOVA, followed by Tukey’s HSD post hoc test. p -Values were considered as significant when * p < 0.05, ** p < 0.01. ( C ) Representative immunoblots showing the levels of cell death, proliferation and cell cycle proteins after TMZ treatment. β-Actin was used as a loading control. WT and RECQL4 KO LN18 were exposed to olaparib for 72 h and analysed for cell viability ( D ) and proliferation ( E ). The results were normalized to control cells (CTRL, with 0.05% DMSO as a solvent) and represent means ± SEM ( n = 4). Statistical significance was calculated as above. ( F ) Representative immunoblots showing the levels of cell death, proliferation and cell cycle proteins after olaparib treatment in WT and RECQL4 KO cells. β-Actin was used as a loading control. ( G ) Cell cycle distribution analysis of WT and RECQL4 KO LN18 cells after TMZ (500 µM) or OLA (10 µM) treatment for 72 h. The results are shown as percentage of cell number in each phase of the cell cycle and represent means ± SEM ( n = 3). Statistical significance was determined by Kruskal–Wallis one-way ANOVA followed by Dunnett’s post hoc test. p -Values were considered as significant when * p < 0.05., ** p < 0.01. ( H) Representative images of immunofluorescent staining showing γH2AX foci and cell nuclei in WT and RECQL4 KO LN18 cells exposed to TMZ (500 µM) or OLA (10 µM) for 48 h. Scale bar represents 10 µm.

Article Snippet: Cells were seeded on coverslips (3 × 10 4 cells/well) for 30 h. The medium was changed to PBS (with Ca 2+ and Mg 2+ ) to induce starvation conditions and the cells were cultured for 18 h. To visualize mitochondrial network, cells were stained with 250 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in dark at 37 °C for 20 min, washed 3 times with a complete medium and 3 times with a PBS, fixed with 4% PFA/PBS pH 7.4 for 15 min and permeabilised with 100% methanol for 10 min. Unspecific signals were blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in 0.3% Triton X-100/PBS for 40 min at RT, followed by incubation with anti-RECQL4 Ab (1:200, Novus Biological) in 3% NDS, 1% BSA in 0.3% Triton X-100/PBS at +4 °C overnight.

Techniques: Western Blot, Control, Solvent, Staining

Journal: Cancers

Article Title: Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

doi: 10.3390/cancers12102919

Figure Lengend Snippet: RECQL4 depletion does not sensitize LN229 glioma cells to temozolomide or olaparib. Analyses of viability and proliferation of WT and RECQL4-deficient LN229 cells exposed for 72 h to TMZ (250 and 500 µM) ( A,B ) or olaparib (5 and 10 µM) ( D,E ). The results were normalized to control cells (CTRL) and represent means ± SEM ( n =4). Statistical significance was determined by RM two-way ANOVA, with followed by Tukey’s HSD post hoc test. Representative immunoblots show the levels of cell death, proliferation and cell cycle proteins after the treatments of WT and RECQL4 KO LN229 cells with TMZ ( C ) or olaparib ( F ). β-Actin was used as a loading control.

Article Snippet: Cells were seeded on coverslips (3 × 10 4 cells/well) for 30 h. The medium was changed to PBS (with Ca 2+ and Mg 2+ ) to induce starvation conditions and the cells were cultured for 18 h. To visualize mitochondrial network, cells were stained with 250 nM MitoTracker CMXRos (Invitrogen, San Diego, CA, USA) in dark at 37 °C for 20 min, washed 3 times with a complete medium and 3 times with a PBS, fixed with 4% PFA/PBS pH 7.4 for 15 min and permeabilised with 100% methanol for 10 min. Unspecific signals were blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in 0.3% Triton X-100/PBS for 40 min at RT, followed by incubation with anti-RECQL4 Ab (1:200, Novus Biological) in 3% NDS, 1% BSA in 0.3% Triton X-100/PBS at +4 °C overnight.

Techniques: Control, Western Blot