human medulloblastoma cell lines daoy  (ATCC)

Journal: bioRxiv

Article Title: Modulation of Stemness and Differentiation Regulators by Valproic Acid in Medulloblastoma

doi: 10.1101/2024.09.23.614476

Figure Lengend Snippet: VPA reduces MB cell viability. A D283 and Daoy human MB cells were treated with a range of VPA concentrations (0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 mM) for 48 h and cell viability was measured by a trypan exclusion assay. IC 50 concentrations of VPA for MB cells with 95% confidence interval (CI) was 2.3 mM for D283 and 2.2 mM for Daoy cells. B Immunofluorescence assay using antibodies against H3K9ac, histone H3 and nuclei marker (DAPI) in MB control and VPA-treated cells. The IC 50 concentration of VPA (2.3 mM for D283 and 2.2 mM for Daoy) was used. Semi-quantification of H3K9ac signal intensity relative to DAPI is shown. Results are mean ± SD of three independent experiments; * P < 0.05 and **** P < 0.0001 compared to controls.

Article Snippet: Data available in the Human Protein Atlas database ( http://proteinatlas.org ) confirms different MYC expression in D283 and Daoy cells [ ].

Techniques: Exclusion Assay, Immunofluorescence, Marker, Control, Concentration Assay

Journal: bioRxiv

Article Title: Modulation of Stemness and Differentiation Regulators by Valproic Acid in Medulloblastoma

doi: 10.1101/2024.09.23.614476

Figure Lengend Snippet: VPA leads to cell cycle arrest in MB cells. A Cell cycle analysis of D283 and Daoy cells treated with VPA and controls. B Relative mRNA levels of CDKN1A and MYC in MB cells were assessed using RT-qPCR. C Western blot analysis of the p21 protein in MB cells after VPA exposure. Relative Densitometric Unit (RDU) analysis normalized by β-actin and corrected by control is shown. All experiments were conducted using the IC 50 concentration of VPA (2.3 mM for D283 and 2.2 mM for Daoy) for 48 h. Results are mean ± SD of three independent experiments; * P < 0.05 and **** P < 0.0001 compared to controls.

Article Snippet: Data available in the Human Protein Atlas database ( http://proteinatlas.org ) confirms different MYC expression in D283 and Daoy cells [ ].

Techniques: Cell Cycle Assay, Quantitative RT-PCR, Western Blot, Control, Concentration Assay

Journal: bioRxiv

Article Title: Modulation of Stemness and Differentiation Regulators by Valproic Acid in Medulloblastoma

doi: 10.1101/2024.09.23.614476

Figure Lengend Snippet: VPA impairs expansion of MB neurospheres enriched with stemness marker genes. A Representative images of D283 and Daoy MB cells and derived neurospheres. B Relative mRNA levels of SOX2, NES and PRTG in monolayer cells and spheres were verified using RT-qPCR. C VPA effects on MB sphere formation after 5 days of drug exposure. Semi-quantitative analysis of sphere size in VPA-treated and control spheres is shown. D After 5 days of MB neurosphere growth, VPA was added and sphere size and number were evaluated after 48 h. Semi-quantitative analyses of sphere size and number in VPA-treated and control spheres are shown. Images were taken in an inverted microscope with 5 X magnification and the scale bar corresponds to 500 μm. E Immunofluorescence assay against H3K9ac, histone H3 and nuclei marker (DAPI) was performed in VPA-treated and control spheres. Fluorescent images were taken in an inverted microscope with 20 X magnification and the scale bar corresponds to 100 μm. F. Relative mRNA levels of SOX2 , NES, and PRTG in control and VPA-treated neurospheres were verified using RT-qPCR. Experiments were conducted using the IC 50 concentration (2.3 mM for D283 and 2.2 mM for Daoy) of VPA. Results represent the mean ± SD of three independent experiments; * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 compared to controls.

Article Snippet: Data available in the Human Protein Atlas database ( http://proteinatlas.org ) confirms different MYC expression in D283 and Daoy cells [ ].

Techniques: Marker, Derivative Assay, Quantitative RT-PCR, Control, Inverted Microscopy, Immunofluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Modulation of Stemness and Differentiation Regulators by Valproic Acid in Medulloblastoma

doi: 10.1101/2024.09.23.614476

Figure Lengend Snippet: VPA increases CDKN1A and TP53 while reducing MYC in MB-derived neurospheres. A-B Relative mRNA levels of CDKN1A , MYC and TP53 in MB neurospheres were verified using RT-qPCR. C ChIP-qPCR for the promoter region of CDKN1A and TP53 in D283-derived spheres. Experiments were conducted using the IC 50 concentration (2.3 mM for D283 and 2.2 mM for Daoy) of VPA for 48 h. Results are mean ± SD of three independent experiments; * P < 0.05, ** P < 0.01, and **** P < 0.0001 compared to controls. For ChIP results * P < 0.05 and ** P < 0.01 comparing VPA-treated cells versus controls immunoprecipitated with anti-IgG or anti-H3K9ac; # P < 0.05 comparing samples immunoprecipitated with the same antibody (IgG versus H3K9ac) between VPA-treated and control groups.

Article Snippet: Data available in the Human Protein Atlas database ( http://proteinatlas.org ) confirms different MYC expression in D283 and Daoy cells [ ].

Techniques: Derivative Assay, Quantitative RT-PCR, Concentration Assay, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: Modulation of Stemness and Differentiation Regulators by Valproic Acid in Medulloblastoma

doi: 10.1101/2024.09.23.614476

Figure Lengend Snippet: VPA effects on features of neuronal differentiation in MB cells. A Representative images of morphological changes in VPA-treated and control D283 and Daoy MB cells. B Relative mRNA levels of TUBB3 and ENO2 in MB cells were verified using RT-qPCR. C Immunofluorescence assay against neuronal differentiation marker protein beta III tubulin and nuclei marker (DAPI). D Relative mRNA levels of TUBB3 and ENO2 in VPA-treated and control neurospheres derived from either D283 or Daoy cells were measured using RT-qPCR. E Immunofluorescence assay against neuronal differentiation marker beta III tubulin and nuclei marker (DAPI) in VPA-treated and control neurospheres. The IC 50 concentration of VPA (2.3 mM for D283 and 2.2 mM for Daoy) was used for treatment. Fluorescent images were taken in an inverted microscope with a magnification of 10 X (monolayer) or 20 X (neurospheres). Results are mean ± SD of three independent experiments; ** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared to controls.

Article Snippet: Data available in the Human Protein Atlas database ( http://proteinatlas.org ) confirms different MYC expression in D283 and Daoy cells [ ].

Techniques: Control, Quantitative RT-PCR, Immunofluorescence, Marker, Derivative Assay, Concentration Assay, Inverted Microscopy