Journal: Cell Death & Disease

Article Title: Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell

doi: 10.1038/s41419-023-05611-8

Figure Lengend Snippet: A The percentage of drugs showing synergistic activity with 8-CPT-cAMP in agents grouped by targets. B Morphology alteration of rat malignant glioma C6 cells and human glioblastoma U87MG cells treated with CM for 2 days. V: Vehicle (1‰ DMSO); C: 0.5 mM 8-CPT-cAMP; M: 1 μM MS275. C The fold change of neuronal genes in the C, M, CM-treated group compared with the control group. D Immunofluorescence analysis of MAP2 (upper, red) and TUBB3 (down, red) in U87MG cells treated with CM for 2 days. The nucleus was stained with DAPI (green). E The percentage of MAP2-positive cells exposure to different regimens of CM. C: +0.125 mM, ++ 0.25 mM, +++ 0.5 mM; M: +0.25 μM, ++ 0.5 μM, +++ 1 μM. Data are shown as mean ± SD ( n = 3 per group). Compared to vehicle control, one-way ANOVA with Dunnett post-test was used. * P < 0.05; ** P < 0.01; *** P < 0.001. F Western blot analysis of MAP2/TUBB3/GFAP/PCNA in CM-treated U87MG cells for 2 days. Tubulin was used as a loading control. G Flow cytometry analysis of the MAP2/TUBB3 in U87MG cells treated with CM for 2 days for 3 and 10 days. The red numeric in the upper right corner represents the percentages (%) of MAP2-positive and TUBB3-positive cells in all tested cells.

Article Snippet: C6 cells were treated with 0.5 mM 8-CPT-cAMP (Enzo life sciences, USA) in the presence or absence of one agent in 349 drugs (Selleck, USA) [ ], and then subject to morphologic evaluation.

Techniques: Activity Assay, Immunofluorescence, Staining, Western Blot, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell

doi: 10.1038/s41419-023-05611-8

Figure Lengend Snippet: A Morphology alteration of U87MG cells after drug treatment and drug withdrawal +: drug treatment; −: drug withdrawal. The cells were treated for 3 days, followed by 3 days withdrawal or treated for 6 days, followed by 6 days withdrawal. B Flow cytometry analysis of EdU staining (green) in U87MG cells with different combinations of CM for 72 h. C The percentage of EdU-positive cells in groups treated with V, C, M, CM for 3 days. Mean ± SD is shown in the bar plot ( n = 3 per group). Compared to vehicle control, One-way ANOVA with Dunnett post-test was used. ** P < 0.01; *** P < 0.001. D Representative trace of fast inward currents recorded in voltage-clamp mode. E Voltage-gated Na + current amplitude of U87MG cells treated with V or CM for 2 days ( n = 7 per group). Cells were depolarized from −80 mV to 60 mV in 10 mV increments. F Na + currents in U87MG cells treated with V or CM for 2 days in the presence or absence of tetrodotoxin (TTX) ( n = 7 per group). G , H Free-tumor survival of mice after inoculation of treated U87MG cells and tumor volume on the 27th day. U87MG cells were treated with V or CM for 72 h, and then 2 × 10 6 cells were injected subcutaneously in BALB/c-nu/nu mice ( n = 5). Tumor greater than 30 mm 3 was considered as developed one and free-tumor survival was presented as Kaplan–Meier survival curve. The log-rank test was used for comparing V and CM. * P <0.05. V: Vehicle (1‰ DMSO); C: 0.5 mM 8-CPT-cAMP; M: 1 μM MS275.

Article Snippet: C6 cells were treated with 0.5 mM 8-CPT-cAMP (Enzo life sciences, USA) in the presence or absence of one agent in 349 drugs (Selleck, USA) [ ], and then subject to morphologic evaluation.

Techniques: Flow Cytometry, Staining, Injection

Journal: Cell Death & Disease

Article Title: Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell

doi: 10.1038/s41419-023-05611-8

Figure Lengend Snippet: A Volcano plot showing the changes in gene expression of U87MG cells treated with V or CM for 2 days. The gene symbols with orange/blue represent typical genes related to neuron-differentiation/oncogenesis. B Gene set enrichment analysis (GSEA) comparing V- and CM-treated U87MG cells. C The heatmap of specific neuron biomarkers in V- and CM-treated U87MG cells. D , E GSEA analysis of cell cycle/undifferentiation gene sets ( D ) and neuron marker/neurotransmitter gene sets ( E ). P < 0.05 and FDR < 0.05 were considered as significant. F Single sample GSEA (ssGSEA) analysis of V-, C-, M-, and CM-treated U87MG cells. Kruskal–Wallis test was used to determine the significance of differences between groups. G Heatmap of gene sets of undifferentiation-related genes and neuron differentiation-related genes. V, Vehicle (1‰ DMSO); C, 0.5 mM 8-CPT-cAMP; M, 1 μM MS275.

Article Snippet: C6 cells were treated with 0.5 mM 8-CPT-cAMP (Enzo life sciences, USA) in the presence or absence of one agent in 349 drugs (Selleck, USA) [ ], and then subject to morphologic evaluation.

Techniques: Expressing, Marker

Journal: Cell Death & Disease

Article Title: Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell

doi: 10.1038/s41419-023-05611-8

Figure Lengend Snippet: A Average profile of H3K9ac/H3K14ac peaks binding to TSS region in U87MG cells treated with V or CM for 2 days. B Dysregulated genes with H3K9ac/H3K14ac tags identified by ChIP-seq. C Gene enrichment analysis of genes with altered H3K9ac/H3K14ac tags. Red trace lines indicate adjusted P = 0.05. D Venn diagrams showing gene counts regulated by H3K9ac and/or H3K14ac. E , F Heatmap of genes with altered H3K9ac/H3K14ac tags. G , H Representative peak plot of oncogenes regulated by H3K9ac (MCM1 and MYC). H Representative peak plot of neuron development-related genes regulated by H3K9ac/H3K14ac (GNAQ1 and BMP7). V, Vehicle (1‰ DMSO); C, 0.5 mM 8-CPT-cAMP; M, 1 μM MS275.

Article Snippet: C6 cells were treated with 0.5 mM 8-CPT-cAMP (Enzo life sciences, USA) in the presence or absence of one agent in 349 drugs (Selleck, USA) [ ], and then subject to morphologic evaluation.

Techniques: Binding Assay, ChIP-sequencing

Journal: Cell Death & Disease

Article Title: Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell

doi: 10.1038/s41419-023-05611-8

Figure Lengend Snippet: A Morphology alteration of human GSCs (GSC-1 and GSC-11 cells) treated with V, C, M, CM for 48 h. Pictures were taken at ×20 magnification. Scale bars, 50 μm. B Quantification of EdU-positive cells in GSC-1 and GSC-11 treated with V, C, M, CM for 48 h. C Flow cytometry analysis for testing the protein levels of MAP2/TUBB3 in GSC-1 and GSC-11 cells treated with V, C, M, CM for 48 h. D Western blot analysis for testing H3K9/K14ac and CD133 expression in GSC-1 (left) and GSC-11 cells (right) treated with V, C, M, CM for 48 h. E Immunofluorescent analysis for voltage-gated sodium channels (NaV) in GSC-1 and GSC-11 treated with CM for 72 h. Pictures were taken at ×40 magnification. Scale bars, 25 μm. F , G GO analysis after RNA-seq and heatmaps of dysregulated genes for GSC-11cells treated with V and CM for 48 h. Blue: upregulated genes (CM vs V), red: downregulated genes (CM vs V). Red lines indicate adjusted P = 0.05. H Gene enrichment analysis of genes with altered H3K9ac/H3K14ac tags after ChIP-seq in GSC-11 cells treated with V and CM for 48 h. Red trace lines indicate adjusted P = 0.05. All data was compared to vehicle with one-way ANOVA. n.s., not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001. Mean ± SD is shown in the bar plot ( n = 3 per group). V, Vehicle (1‰ DMSO); C, 0.5 mM 8-CPT-cAMP; M, 1 μM MS275.

Article Snippet: C6 cells were treated with 0.5 mM 8-CPT-cAMP (Enzo life sciences, USA) in the presence or absence of one agent in 349 drugs (Selleck, USA) [ ], and then subject to morphologic evaluation.

Techniques: Flow Cytometry, Western Blot, Expressing, RNA Sequencing Assay, ChIP-sequencing

Journal: PLoS ONE

Article Title: Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

doi: 10.1371/journal.pone.0121551

Figure Lengend Snippet: (a) Treatment with a cGMP agonist (8-Br-cGMP, 1 μM) or PKG agonist (8-pCPT-cGMP, 1 μM) alone. The cells were pretreated with a cGMP inhibitor (8-Br-cAMP, 1 μM), PKG inhibitor (8-CPT-cAMP, 1 μM), Rho-specific inhibitor, (Rhosin, 30 μM), CDC42 inhibitor (ZCL278, 30 μM) or Rac1 inhibitor (Z62954982, 50 μM) for 2 hours, followed by the addition of SNP at a final concentration of 10 μM and incubation for another 24 hours. (b) The results are expressed as the means ± SD of 3 experiments.

Article Snippet: In some cases, the cells were first pretreated for 2 hours with a cGMP inhibitor (8-Br-cAMP, 1 μM, Proteinkinase, Germany), PKG inhibitor (8-CPT-cAMP, 1 μM, Proteinkinase, Germany), Rho-specific inhibitor, (Rhosin, 30 μM, Calbiochem, USA), CDC42 inhibitor (ZCL278, 30 μM, Calbiochem, USA) or Rac1 inhibitor (Z62954982, 50 μM, Calbiochem, USA).

Techniques: Concentration Assay, Incubation

Journal: PLoS ONE

Article Title: Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

doi: 10.1371/journal.pone.0121551

Figure Lengend Snippet: Treatment with a cGMP agonist (8-Br-cGMP, 1 μM) or PKG agonist (8-pCPT-cGMP, 1 μM) alone was able to enhance F-actin reorganization in HaCaT cells. Additionally, the cells were pretreated with a cGMP inhibitor (8-Br-cAMP, 1 μM), PKG inhibitor (8-CPT-cAMP, 1 μM), Rho-specific inhibitor (Rhosin, 30 μM), CDC42 inhibitor (ZCL278, 30 μM) or Rac1 inhibitor (Z62954982, 50 μM) for 2 hours, followed by the addition of SNP at a final concentration of 10 μM and incubation for another 24 hours. The percentage of cells exhibiting stress fibres (mean ± SD, n = 3 slides) in a total population of 100 cells was calculated for each slide.

Article Snippet: In some cases, the cells were first pretreated for 2 hours with a cGMP inhibitor (8-Br-cAMP, 1 μM, Proteinkinase, Germany), PKG inhibitor (8-CPT-cAMP, 1 μM, Proteinkinase, Germany), Rho-specific inhibitor, (Rhosin, 30 μM, Calbiochem, USA), CDC42 inhibitor (ZCL278, 30 μM, Calbiochem, USA) or Rac1 inhibitor (Z62954982, 50 μM, Calbiochem, USA).

Techniques: Concentration Assay, Incubation

Journal: PLoS ONE

Article Title: Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

doi: 10.1371/journal.pone.0121551

Figure Lengend Snippet: Treatment with 10 μM SNP, a cGMP agonist (8-Br-cGMP, 1 μM) or a PKG agonist (8-pCPT-cGMP, 1 μM) alone was used to stimulate HaCaT cells, or the cells were pretreated with a cGMP inhibitor (8-Br-cAMP, 1 μM) or PKG inhibitor (8-CPT-cAMP, 1 μM) for 2 hours, followed by the addition of SNP at a final concentration of 10 μM and incubation for another 24 hours. (a) The mRNA expression of CDC42, Rac1 and RhoA was quantified via semi-quantitative RT-PCR. (b) The relative levels of CDC42, Rac1 and RhoA mRNA expression were determined by comparison with the internal reference GAPDH.

Article Snippet: In some cases, the cells were first pretreated for 2 hours with a cGMP inhibitor (8-Br-cAMP, 1 μM, Proteinkinase, Germany), PKG inhibitor (8-CPT-cAMP, 1 μM, Proteinkinase, Germany), Rho-specific inhibitor, (Rhosin, 30 μM, Calbiochem, USA), CDC42 inhibitor (ZCL278, 30 μM, Calbiochem, USA) or Rac1 inhibitor (Z62954982, 50 μM, Calbiochem, USA).

Techniques: Concentration Assay, Incubation, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

doi: 10.1371/journal.pone.0121551

Figure Lengend Snippet: HaCaT cells were treated with SNP (10 μM), a cGMP agonist (8-Br-cGMP, 1 μM) or a PKG agonist (8-pCPT-cGMP, 1 μM) alone for 10 minutes, or the cells were pretreated with a cGMP inhibitor or PKG inhibitor for 10 minutes, then stimulated with 10 μM SNP for 10 minutes. Finally, the total or active proteins of CDC42, Rac1 and RhoA were quantified in pull-down assays (a). The relative ratios of the active CDC42, Rac1 and RhoA proteins against the internal reference β-actin are shown in panels (b).

Article Snippet: In some cases, the cells were first pretreated for 2 hours with a cGMP inhibitor (8-Br-cAMP, 1 μM, Proteinkinase, Germany), PKG inhibitor (8-CPT-cAMP, 1 μM, Proteinkinase, Germany), Rho-specific inhibitor, (Rhosin, 30 μM, Calbiochem, USA), CDC42 inhibitor (ZCL278, 30 μM, Calbiochem, USA) or Rac1 inhibitor (Z62954982, 50 μM, Calbiochem, USA).

Techniques: