Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: DNMT3A and DNMT3B expression levels. Transcript levels (left panels) and protein expression (right panels) of DNMT3A ( a ) and DNMT3B ( b ) in RD cells transfected with si-DNMT3A, si-DNMT3B or si-NC 48 h before being exposed or not to radiation (4 Gy). q-PCR analysis was expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1, and GAPDH was used as endogenous control. Bars represent mean values ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; **, p < 0.01 vs. si-NC/0 Gy; $$$, p < 0.001 vs. si-NC/4 Gy. For Western blotting assay, tubulin was used as loading control. In are reported the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Expressing, Transfection, Control, Western Blot

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: DNMT3A and DNMT3B knocking down increase sensitivity of RMS cells to radiation. ( a ) Representative images of RD cells stained with Giemsa and photographed at 20× magnification at 144 h after DNMT3A or DNMT3B depletion and 96 h after irradiation. si-DNMT3A samples showed flattened, enlarged, and irregular morphology, whilst si-DNMT3B cells displayed more elongated cellular bodies. Scale bar 200 μM. ( b ) RD cell proliferation assessed by trypan blue exclusion staining at 144 h after silencing with specific siRNA and irradiated or not with a single dose of 4 Gy. Results represent mean values ± SD of four independent experiments expressed as fold increase over negative control (si-NC/0 Gy), arbitrarily set at 1. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; **, p < 0.01 vs. si-NC/0 Gy; #, p < 0.05 vs. si-DNMT3B/0 Gy; $$, p < 0.01; $, p < 0.05 vs. si-NC/4 Gy. ( c ) Western blotting showed γ-H2AX expression levels in RD cells transfected with si-DNMT3A, si-DNMT3B or si-NC at 3 h after radiation treatment. Tubulin expression was used as loading control. ( d ) Western blotting assay of specific proteins involved in DNA damage signaling and response at 24 h after irradiation. Tubulin was used as internal control. ( e ) RD cells after the combined treatment were seeded at low density and allowed to grow for 12 days. Representative pictures of colonies stained with crystal violet. Histograms represent means ± SD of five independent experiments, each performed in triplicate. Colony-forming efficiency, calculated by crystal violet absorbance, was expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001, vs. si-NC/0 Gy; §§§, p < 0.001 vs. si-DNMT3A/0 Gy; ###, p < 0.001 vs. si-DNMT3B/0 Gy; $$$, p < 0.001; $$, p < 0.01 vs. si-NC/4 Gy. reports the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Staining, Irradiation, Negative Control, Western Blot, Expressing, Transfection, Control

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: DNMT3A and DNMT3B depletion induced a marked p21 overexpression in irradiated RD cells. ( a ) mRNA (left panel) and protein levels (right panel) of p21 (CDKN1A) in RD cells after DNMT3A or DNMT3B silencing and IR exposure. q-PCR analysis was expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1 and GAPDH was used as endogenous control. Bars represent means ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; *, p < 0.05 vs. si-NC/0 Gy; $$$, p < 0.001, $, p < 0.05 vs. si-NC/4 Gy. For Western blotting assay, tubulin was used as loading control. ( b ) miR-106b expression levels analysed by q-PCR assay in RD cells after the combined treatment (silencing/irradiation) and expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1. U6 was used as internal control. Histograms represent mean values ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: **, p < 0.01; *, p < 0.05 vs. si-NC/0 Gy; #, p < 0.05 vs. si-DNMT3B/0 Gy. In are reported the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Over Expression, Irradiation, Control, Western Blot, Expressing

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: p38 inhibition counteracts γ-H2AX up-regulation and the reduced clonogenic ability induced by DNMT3A/3B silencing. ( a ) Western blotting showing the increase of phosphorylated p38 (p-p38) in RD cells at 24 h upon irradiation and 72 h after DNMT3A or DNMT3B knocking down. Levels of total p38 was not perturbed by the specific siRNA transfection and/or irradiation. Tubulin expression was used as loading control. ( b ) Western blotting showing γ-H2AX levels in RD cells pre-treated with SB203580 or DMSO, transfected with si-DNMT3A, si-DNMT3B or si-NC, and exposed or not to radiation. Tubulin expression was used as internal control. ( c ) RD cells at 24 h after irradiation were seeded at low density and allowed to grow for 10 days. Representative images of colonies stained with crystal violet. Bars represent means ± SD of three independent experiments, each performed in triplicate. Colony forming efficiency, calculated by crystal violet absorbance, was expressed as fold increase over si-NC/DMSO/0 Gy (left panel) and si-NC/DMSO/4 Gy (right panel) arbitrarily set at 1. Statistical analyses were performed by using two-way ANOVA: **, p < 0.01, vs. si-NC/DMSO/0 Gy; §, p < 0.05 vs. si-DNMT3A/DMSO/0 Gy; ##, p < 0.01 vs. si-DNMT3B/DMSO/0 Gy (left panel); ***, p < 0.001, vs. si-NC/DMSO/4 Gy; §, p < 0.05 vs. si-DNMT3A/DMSO/4 Gy; #, p < 0.05 vs. si-DNMT3B/DMSO/4 Gy (right panel). presents the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Inhibition, Western Blot, Irradiation, Transfection, Expressing, Control, Staining

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: Morphological changes induced by DNMT3A and DNMT3B silencing and radiation treatment in RD cells. ( a ) IF assays showing the expression of Phalloidin in si-DNMT3A and si-DNMT3B depleted RD cells, also exposed or not to IR. DAPI was used for nuclear staining. Images were acquired by using ApoTome microscope at 40× magnification. Scale bar 50 μm. ( b ) Histograms represent the mean value ± SD of nucleus (N)/cytoplasm (C) area (left panel) or perimeter (right panel) ratio calculated by using ImageJ software. Area and perimeter of N and C were measured for each cell in four random merged Phalloidin/DAPI images for the specific silencing (**, p < 0.01; ***, p < 0.001 vs. si-NC/0 Gy). In multinucleate cells, the nuclear area or perimeter was calculated as the sum of each nucleus.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Expressing, Staining, Microscopy, Software

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: Radiotherapy maintains the myogenic program induced by DNMT3B knocking down. ( a ) Quantitative analysis of differentiation marker MYOD, MYOGENIN and MYHC in RD cells transfected with si-DNMT3A or si-DNMT3B and then exposed to 4 Gy. Data were expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1 and GAPDH was used as endogenous control. Bars represent mean values ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: **, p < 0.01; *, p < 0.05 vs. si-NC/0 Gy; $$, p < 0.01, $, p < 0.05 vs. si-NC/4 Gy. ( b ) q-PCR assay of myomiR miR-133a and miR-206 in RD cells at 144 h after transfection with si-DNMT3A or si-DNMT3B and 96 h after radiation treatment. Data were expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1. U6 was used as internal control. Bars represent means ± SD of five independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; **, p < 0.01 vs. si-NC/0 Gy; ###, p < 0.001, #, p < 0.05 vs. si-DNMT3B/0 Gy; $$$, p < 0.001, $, p < 0.05 vs. si-NC/4 Gy. ( c ) Western blotting assay showing MyoD, Myogenin, MyHC and γ-H2AX up-regulation in RD cells transfected with si-DNMT3B (144 h) and exposed to IR (96 h). Tubulin expression was used as loading control. presents the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Marker, Transfection, Control, Western Blot, Expressing

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: DNMT3A and DNMT3B expression levels. Transcript levels (left panels) and protein expression (right panels) of DNMT3A ( a ) and DNMT3B ( b ) in RD cells transfected with si-DNMT3A, si-DNMT3B or si-NC 48 h before being exposed or not to radiation (4 Gy). q-PCR analysis was expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1, and GAPDH was used as endogenous control. Bars represent mean values ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; **, p < 0.01 vs. si-NC/0 Gy; $$$, p < 0.001 vs. si-NC/4 Gy. For Western blotting assay, tubulin was used as loading control. In are reported the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Expressing, Transfection, Control, Western Blot

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: DNMT3A and DNMT3B knocking down increase sensitivity of RMS cells to radiation. ( a ) Representative images of RD cells stained with Giemsa and photographed at 20× magnification at 144 h after DNMT3A or DNMT3B depletion and 96 h after irradiation. si-DNMT3A samples showed flattened, enlarged, and irregular morphology, whilst si-DNMT3B cells displayed more elongated cellular bodies. Scale bar 200 μM. ( b ) RD cell proliferation assessed by trypan blue exclusion staining at 144 h after silencing with specific siRNA and irradiated or not with a single dose of 4 Gy. Results represent mean values ± SD of four independent experiments expressed as fold increase over negative control (si-NC/0 Gy), arbitrarily set at 1. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; **, p < 0.01 vs. si-NC/0 Gy; #, p < 0.05 vs. si-DNMT3B/0 Gy; $$, p < 0.01; $, p < 0.05 vs. si-NC/4 Gy. ( c ) Western blotting showed γ-H2AX expression levels in RD cells transfected with si-DNMT3A, si-DNMT3B or si-NC at 3 h after radiation treatment. Tubulin expression was used as loading control. ( d ) Western blotting assay of specific proteins involved in DNA damage signaling and response at 24 h after irradiation. Tubulin was used as internal control. ( e ) RD cells after the combined treatment were seeded at low density and allowed to grow for 12 days. Representative pictures of colonies stained with crystal violet. Histograms represent means ± SD of five independent experiments, each performed in triplicate. Colony-forming efficiency, calculated by crystal violet absorbance, was expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001, vs. si-NC/0 Gy; §§§, p < 0.001 vs. si-DNMT3A/0 Gy; ###, p < 0.001 vs. si-DNMT3B/0 Gy; $$$, p < 0.001; $$, p < 0.01 vs. si-NC/4 Gy. reports the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Staining, Irradiation, Negative Control, Western Blot, Expressing, Transfection, Control

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: Cell cycle distribution of RD cells after DNMT3A and DNMT3B silencing and IR exposure. ( a ) Representative cell cycle distribution of RD cells at 24 h after irradiation and 72 h upon DNMT knocking down with the specific siRNAs. ( b ) Flow-cytometry assay-related bars showing cell percentage in G1, S, and G2 phases in RD cells silenced for DNMT3A or DNMT3B and exposed to radiation. Data are average value of four independent experiments. ( c ) Western blotting assays showing cyclin B1 and cyclin D1 levels in RD cells with or without specific siRNAs and IR. Tubulin was used as internal control. In are reported the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Irradiation, Flow Cytometry, Western Blot, Control

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: DNMT3A and DNMT3B depletion induced a marked p21 overexpression in irradiated RD cells. ( a ) mRNA (left panel) and protein levels (right panel) of p21 (CDKN1A) in RD cells after DNMT3A or DNMT3B silencing and IR exposure. q-PCR analysis was expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1 and GAPDH was used as endogenous control. Bars represent means ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; *, p < 0.05 vs. si-NC/0 Gy; $$$, p < 0.001, $, p < 0.05 vs. si-NC/4 Gy. For Western blotting assay, tubulin was used as loading control. ( b ) miR-106b expression levels analysed by q-PCR assay in RD cells after the combined treatment (silencing/irradiation) and expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1. U6 was used as internal control. Histograms represent mean values ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: **, p < 0.01; *, p < 0.05 vs. si-NC/0 Gy; #, p < 0.05 vs. si-DNMT3B/0 Gy. In are reported the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Over Expression, Irradiation, Control, Western Blot, Expressing

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: p38 inhibition counteracts γ-H2AX up-regulation and the reduced clonogenic ability induced by DNMT3A/3B silencing. ( a ) Western blotting showing the increase of phosphorylated p38 (p-p38) in RD cells at 24 h upon irradiation and 72 h after DNMT3A or DNMT3B knocking down. Levels of total p38 was not perturbed by the specific siRNA transfection and/or irradiation. Tubulin expression was used as loading control. ( b ) Western blotting showing γ-H2AX levels in RD cells pre-treated with SB203580 or DMSO, transfected with si-DNMT3A, si-DNMT3B or si-NC, and exposed or not to radiation. Tubulin expression was used as internal control. ( c ) RD cells at 24 h after irradiation were seeded at low density and allowed to grow for 10 days. Representative images of colonies stained with crystal violet. Bars represent means ± SD of three independent experiments, each performed in triplicate. Colony forming efficiency, calculated by crystal violet absorbance, was expressed as fold increase over si-NC/DMSO/0 Gy (left panel) and si-NC/DMSO/4 Gy (right panel) arbitrarily set at 1. Statistical analyses were performed by using two-way ANOVA: **, p < 0.01, vs. si-NC/DMSO/0 Gy; §, p < 0.05 vs. si-DNMT3A/DMSO/0 Gy; ##, p < 0.01 vs. si-DNMT3B/DMSO/0 Gy (left panel); ***, p < 0.001, vs. si-NC/DMSO/4 Gy; §, p < 0.05 vs. si-DNMT3A/DMSO/4 Gy; #, p < 0.05 vs. si-DNMT3B/DMSO/4 Gy (right panel). presents the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Inhibition, Western Blot, Irradiation, Transfection, Expressing, Control, Staining

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: DNMT3A silencing radiosensitizes RD cells by triggering p16-related senescence program. ( a ) Western blotting showing p16 up-regulation in si-DNMT3A RD cells. Tubulin was used as loading control. ( b ) Western blotting assay of specific proteins modulated during the senescence process. Tubulin was used as internal control. ( c ) IF assays showing the expression of Lamin B1 in si-DNMT3A and si-DNMT3B depleted RD cells, also exposed or not to IR. DAPI was used for nuclear staining. Images were acquired by using ApoTome microscope at 40× magnification. Scale bar 20 μm. presents the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Western Blot, Control, Expressing, Staining, Microscopy

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: Morphological changes induced by DNMT3A and DNMT3B silencing and radiation treatment in RD cells. ( a ) IF assays showing the expression of Phalloidin in si-DNMT3A and si-DNMT3B depleted RD cells, also exposed or not to IR. DAPI was used for nuclear staining. Images were acquired by using ApoTome microscope at 40× magnification. Scale bar 50 μm. ( b ) Histograms represent the mean value ± SD of nucleus (N)/cytoplasm (C) area (left panel) or perimeter (right panel) ratio calculated by using ImageJ software. Area and perimeter of N and C were measured for each cell in four random merged Phalloidin/DAPI images for the specific silencing (**, p < 0.01; ***, p < 0.001 vs. si-NC/0 Gy). In multinucleate cells, the nuclear area or perimeter was calculated as the sum of each nucleus.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Expressing, Staining, Microscopy, Software

Journal: Cells

Article Title: DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma

doi: 10.3390/cells10112956

Figure Lengend Snippet: Radiotherapy maintains the myogenic program induced by DNMT3B knocking down. ( a ) Quantitative analysis of differentiation marker MYOD, MYOGENIN and MYHC in RD cells transfected with si-DNMT3A or si-DNMT3B and then exposed to 4 Gy. Data were expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1 and GAPDH was used as endogenous control. Bars represent mean values ± SD of three independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: **, p < 0.01; *, p < 0.05 vs. si-NC/0 Gy; $$, p < 0.01, $, p < 0.05 vs. si-NC/4 Gy. ( b ) q-PCR assay of myomiR miR-133a and miR-206 in RD cells at 144 h after transfection with si-DNMT3A or si-DNMT3B and 96 h after radiation treatment. Data were expressed as fold increase over si-NC/0 Gy, arbitrarily set at 1. U6 was used as internal control. Bars represent means ± SD of five independent experiments, each performed in triplicate. Statistical analyses were performed by using two-way ANOVA: ***, p < 0.001; **, p < 0.01 vs. si-NC/0 Gy; ###, p < 0.001, #, p < 0.05 vs. si-DNMT3B/0 Gy; $$$, p < 0.001, $, p < 0.05 vs. si-NC/4 Gy. ( c ) Western blotting assay showing MyoD, Myogenin, MyHC and γ-H2AX up-regulation in RD cells transfected with si-DNMT3B (144 h) and exposed to IR (96 h). Tubulin expression was used as loading control. presents the densitometric analyses of the different experiments performed and the corresponding statistical analysis.

Article Snippet: ERMS cells were seeded at 1.5 × 10 5 cells/well in 12-well plates; small interfering RNA (siRNA) against human DNMT3A, DNMT3B or siRNA negative control (si-DNMT3A, sc-37757; si-DNMT3B, sc-37759; si-NC, sc-37007 by Santa Cruz Biotechnology, Dallas, TX, USA) were combined with RNAiMAX (Invitrogen, Carlsbad, CA, USA) at 60 nM final concentration following the manufacturer’s protocol. si-DNMT3A and si-DNMT3B are a pool of 3 target-specific 19–25 nt siRNAs designed to specifically knock down gene expression.

Techniques: Marker, Transfection, Control, Western Blot, Expressing

Journal: American Journal of Translational Research

Article Title: MicroRNA-129-5p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting DNMT3A

doi:

Figure Lengend Snippet: MiR-129-5p directly targets DNMT3A and negatively regulates cell cycle-related proteins. A. Predicted miR-129-5p binding sites in the 3’-UTR of the DNMT3A gene. B. The expression levels of DNMT3A in non-tumor brain tissues (NBTs) and glioma specimens were determined by western blotting; the fold changes were normalized to β-Actin. The NBTs (n=9) were collected from brain trauma surgery. The glioblastoma (GBM) tissues (n=17) were collected from Surgical specimens of patients with primary glioblastoma. Data represent the means ± SD from three independent experiments. (***P<0.001). C. Pearson’s correlation analysis of the relationship between the relative expression levels of miR-129-5p and the relative protein levels of DNMT3A in Clinical tissue samples. D. Wild-type and mutant DNMT3A 3’-UTR reporter constructs. E. Luciferase reporter assays were performed in U87 and LN229 cells co-transfected with the indicated wild-type or mutant 3’-UTR constructs and the miR-129-5p mimic. The data shown are representative of three independent experiments. Data shown are mean ± SD of three independent experiments. (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: Small interfering RNAs (siRNAs) targeting DNMT3A (sc-37757) was purchased from Santa Cruz Biotechnology (TX, USA).

Techniques: Binding Assay, Expressing, Western Blot, Mutagenesis, Construct, Luciferase, Transfection

Journal: American Journal of Translational Research

Article Title: MicroRNA-129-5p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting DNMT3A

doi:

Figure Lengend Snippet: DNMT3A is highly expressed in gliomas and is associated with cell proliferation. A-D. Levels of DNMT3A were analyzed in low-grade glioma (LGG) and high-grade glioma (HGG) in CGGA, TCGA, GSE4290 and Rembrandt databases. E. Western blotting was applied to detect the level of DNMT3A in clinical tissues, β-Actin was used as the loading control. F, G. Gene positively-associated with DNMT3A expression in the CGGA, Rembrandt, and GSE4290 glioblastoma samples were subjected to gene ontology (GO) analysis and KEEG pathway analysis. Enrichment for biological process in the GO database and KEGG pathways analysis are shown. The orders of the different biological processes is based on their enriched number.

Article Snippet: Small interfering RNAs (siRNAs) targeting DNMT3A (sc-37757) was purchased from Santa Cruz Biotechnology (TX, USA).

Techniques: Western Blot, Control, Expressing

Journal: American Journal of Translational Research

Article Title: MicroRNA-129-5p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting DNMT3A

doi:

Figure Lengend Snippet: Down-regulation of DNMT3A inhibits glioma cell proliferation in vitro. A. U87-MG and LN229 cells were transfected with DNMT3A siRNA and cultured for in 96 h. Cell proliferation was determined using the CCK-8 assay. Data are presented as the means of triplicate experiments. (**P<0.01). B, C. Proliferating cells were examined using the EDU assay. Representative images are shown (original magnification, 200×). (**P<0.01). D, E. The cell cycle phase of U87-MG and LN229 cells transfected with DNMT3A siRNA or negative control (NC) was analyzed by flow cytometry. (**P<0.01). F. A heat map of relative expression of several DNMT3A-associated cell cycle genes in Rembrandt glioblastoma tissues sorted relative to the level of DNMT3A expression (r>0.4). G. Western blot analysis of DNMT3A, CDK2 and cyclinA2 in U87-MG and LN229 cells after knockdown of DNMT3A.

Article Snippet: Small interfering RNAs (siRNAs) targeting DNMT3A (sc-37757) was purchased from Santa Cruz Biotechnology (TX, USA).

Techniques: In Vitro, Transfection, Cell Culture, CCK-8 Assay, EdU Assay, Negative Control, Flow Cytometry, Expressing, Western Blot, Knockdown

Journal: American Journal of Translational Research

Article Title: MicroRNA-129-5p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting DNMT3A

doi:

Figure Lengend Snippet: Reintroduction of DNMT3A reverses the inhibitory effect of miR-129-5p. A. A rescue experiment was performed by introducing pcDNA3.1-DNMT3A or pcDNA3.1 in the presence or absence of ectopic miR-129-5p or miR-ctrl expression in U87-MG and LN229 cells. Western blot assays were performed to detect the levels of DNMT3A, CDK2 and cyclinA2 in the indicated cells. Actin was used as the loading control. B. Cell viability of glioma cells transfected with pcDNA3.1-DNMT3A and miR-129-5p separately or together was detected using the CCK-8 assay. C, D. Cell proliferative potential was evaluated using the EDU assay 48 h after co-transfection of pcDNA3.1-DNMT3A and miR-129-5p. E, F. Cell cycle distribution of glioma cells was measured using flow cytometry.

Article Snippet: Small interfering RNAs (siRNAs) targeting DNMT3A (sc-37757) was purchased from Santa Cruz Biotechnology (TX, USA).

Techniques: Expressing, Western Blot, Control, Transfection, CCK-8 Assay, EdU Assay, Cotransfection, Flow Cytometry

Journal: Cancer Science

Article Title: Micro RNA ‐9 plays a role in interleukin‐10‐mediated expression of E‐cadherin in acute myelogenous leukemia cells

doi: 10.1111/cas.13170

Figure Lengend Snippet: Effect of interleukin‐10 ( IL ‐10) on DNA methyltransferase 3A ( DNMT 3A) expression in leukemia cells. (a) DNMT 3A expression in leukemia cells treated with IL ‐10 was analyzed by real‐time RT ‐ PCR . (b) Western blot analysis. Leukemia cells treated with IL ‐10 were harvested, and subjected to Western blot analysis to monitor the levels of the indicated proteins. (c) Methylation‐specific PCR . Leukemia cells were treated with IL ‐10, and DNA was extracted from the cells. DNA with methylated CpG was processed using a MethylEasy Xceed Rapid DNA Bisulphite Modification Kit. The recovered DNA was amplified by PCR on miR‐9‐3 CpG islands. The experiments were repeated three times. M, methylation of the gene promoter; U, unmethylated gene promoter. (d–f) DNMT 3A expression in MOLM 13 (d), MV 4‐11 (e), and THP ‐1 (f) cells transfected with either control si RNA or DNMT 3A si RNA s was analyzed by real‐time RT ‐ PCR . E‐cadherin expression in leukemia cells transfected with either control or DNMT 3A si RNA s was analyzed by real‐time RT ‐ PCR and flow cytometry. (g) Expression of miR‐9 in leukemia cells transfected with either control or DNMT 3A si RNA s was analyzed using an Mir‐X mi RNA qRT ‐ PCR SYBR Kit to measure the levels of the indicated gene. The results shown represent the mean ± SD of three experiments carried out in triplicate. * P < 0.05; ** P < 0.01.

Article Snippet: A control siRNA and two siRNAs against DNMT3A were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sigma, respectively.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Methylation, Modification, Amplification, Transfection, Flow Cytometry

Journal: Cancer Science

Article Title: Micro RNA ‐9 plays a role in interleukin‐10‐mediated expression of E‐cadherin in acute myelogenous leukemia cells

doi: 10.1111/cas.13170

Figure Lengend Snippet: Effect of interleukin‐10 ( IL ‐10) on leukemia cell engraftment in immunodeficient mice and mouse survival. MOLM 13 cells transduced with control sh RNA or IL ‐10‐specific sh RNA were transplanted into NOD .Cg‐ Rag1 tm1Mom Il2rg tm1Wjl /SzJ mice (control sh RNA mice or IL ‐10 sh RNA mice) by tail vein injections ( n = 10 mice/group). The treatment was initiated after 1 week of transplantation. The mice received either cytarabine (AraC) ( n = 10; 20 mg/kg, 3 days/week for 2 weeks) or PBS ( n = 10; 100 μL, 3 days/week for 2 weeks). (a) Kaplan–Meier analysis of mouse survival ( n = 10). At day 18 (b) and 28 (c) of transplantation, peripheral blood ( PB ) and bone marrow ( BM ) cells were collected and incubated with anti‐ CD 82 antibody. Positive cells were analyzed by flow cytometry ( n = 10). BM cells were collected on day 18 (d) and 28 (f) post‐transplantation and analyzed for IL ‐10, DNA methyltransferase 3A ( DNMT 3A), and E‐cadherin mRNA expression by real‐time RT ‐ PCR ( n = 10). Plasma was collected on day 18 (e) and 28 (g) post‐transplantation and analyzed for micro RNA ‐9 (miR‐9) expression by real‐time RT ‐ PCR ( n = 10). * P < 0.05; ** P < 0.01.

Article Snippet: A control siRNA and two siRNAs against DNMT3A were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sigma, respectively.

Techniques: Transduction, Transplantation Assay, Incubation, Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Micro RNA ‐9 plays a role in interleukin‐10‐mediated expression of E‐cadherin in acute myelogenous leukemia cells

doi: 10.1111/cas.13170

Figure Lengend Snippet: Interleukin‐10 ( IL ‐10) regulates E‐cadherin expression through micro RNA ‐9 (miR‐9). DNMT 3A, DNA methyltransferase 3A; me, methylation.

Article Snippet: A control siRNA and two siRNAs against DNMT3A were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sigma, respectively.

Techniques: Expressing, Methylation

Journal: Cancer Science

Article Title: Micro RNA ‐9 plays a role in interleukin‐10‐mediated expression of E‐cadherin in acute myelogenous leukemia cells

doi: 10.1111/cas.13170

Figure Lengend Snippet: Polymerase chain reaction primers

Article Snippet: A control siRNA and two siRNAs against DNMT3A were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sigma, respectively.

Techniques: Polymerase Chain Reaction

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 3. DNMT3a upregulation is responsible for the hypermethylation of the miR-200c gene promoter. (A) Knockdown of DNMT3a protein in AGS cells following 0, 24 and 48 h of transfection with 100 nM siRNA relative to negative controls. (B) Enhanced expression of DNMT3a protein in the MGC803 cells after stable transfection with the DNMT3a expression vector. (C and D) Bisulfite sequencing PCR showed that DNMT3a knockdown decreased the methyla- tion status of the miR-200c promoter region in the AGS cells, and enhanced DNMT3a expression increased the methylation status of the miR‑200c promoter region in the MGC803 cells. (E) Knockdown of DNMT3a resulted in upregulation of miR-200c expression, whereas enhanced DNMT3a expression resulted in decreased miR-200c expression (*P<0.05, ***P<0.001).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Knockdown, Transfection, Expressing, Stable Transfection, Plasmid Preparation, Methylation Sequencing, Methylation

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 2. Differential expression of DNMT3a in cell lines and tissues. (A) Western blotting showed that the expression of DNMT3a in five GC cell lines was higher than that in immortal gastric epithelial GES-1 cells. (B) Immunohistochemistry assays showed that the expression of DNMT3a in GC tissues was significantly higher compared to that noted in the adjacent non-tumor tissues (magnification, x200).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Immunohistochemistry

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 4. miR-200c suppresses DNMT3a expression and induces endogenous pre-miR-200c and pri-miR-200c reexpression. (A) Putative miR-200c binding sites within the human DNMT3a 3'UTR are shown. The position of the binding sites was numbered relative to the first nucleotide of the 3'UTR. Mutations were introduced into DNMT3a 3'UTR that matched the seed region of miR-200c. (B) Interspecies conservation of putative miR-200c binding sites within the DNMT3a 3'UTR are shown. (C) Transfection with miR-200c reduced DNMT3A protein expression, whereas transfection with miR-200c inhibitors increased DNMT3A protein expression in the SGC7901 (left) and AGS cells (right). (D and E) Relative expression of pre-miR-200c and pri-miR-200c demonstrated by qRT-PCR was increased in the AGS cells following 0, 24 and 48 h transfection with 50 nM miR-200c mimics. (F) Ectopic miR-200c expression inhibits wild-type but not mutant DNMT3A 3'UTR reporter activity in the SGC7901 cells. Cells were co-transfected with miR-200c mimics and with either wild-type or mutant DNMT3A 3'UTR reporter construct. Luciferase activity assay was performed at 48 h after transfection (*P<0.05, ***P<0.001).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Expressing, Binding Assay, Transfection, Quantitative RT-PCR, Mutagenesis, Activity Assay, Construct, Luciferase

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 5. miR-200c mimics and DNMT3a siRNA suppress cell growth. (A and B) SGC7901 and AGS cells transfected with miR-200c mimics showed significantly reduced cellular proliferation compared with the negative control using Cell Counting Kit-8 assay. (C and D) SGC7901 and AGS cells transfected with DNMT3a siRNA also showed significantly reduced cellular proliferation compared with the negative control assaying (*P<0.05).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Transfection, Negative Control, Cell Counting

Journal: Oncology reports

Article Title: Epigenetically deregulated miR-200c is involved in a negative feedback loop with DNMT3a in gastric cancer cells.

doi: 10.3892/or.2016.4996

Figure Lengend Snippet: Figure 6. miR-200c mimics and DNMT3a siRNA suppress cell migration and invasion. (A) Representative images of cell invasion assays showed that miR- 200c mimics suppressed cell invasion in the SGC7901 and AGS cells. (B) Statistical analyses showed that miR-200c mimics suppressed cell invasion in the SGC7901 and AGS cells. (C) Representative images of cell invasion assays showed that DNMT3a siRNA suppressed cell invasion in the SGC7901 and AGS cells. (D) Statistical analyses showed that DNMT3a siRNA suppressed cell invasion in the SGC7901 and AGS cells. (E) Representative images of wound- healing assays showed that miR-200c mimics suppressed cell migration in the SGC7901 and AGS cells. (F) Statistical analyses showed that miR-200c mimics suppressed cell migration in the SGC7901 and AGS cells. (G) Representative images of wound-healing assays showed that DNMT3a siRNA suppressed cell migration in the SGC7901 and AGS cells. (H) Statistical analyses showed that DNMT3a siRNA suppressed cell migration in the SGC7901 and AGS cells (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: A total of 100 nM of siRNA against DNMT3a (sc-37757; Santa Cruz biotechnology, Santa Cruz, CA, USA), double-stranded miR-200c mimics (50 nM), single-stranded miR-200c inhibitor (100 nM) (Ribobio, Guangzhou, China) or their relative negative control RNAs were introduced into cells.

Techniques: Migration

Journal: TURKISH JOURNAL OF BIOLOGY

Article Title: The effects of DNA methyl transferases on antiaging klotho gene expression

doi: 10.3906/biy-1508-36

Figure Lengend Snippet: Figure 1. Genomic localization and structure of DNMT3A (locusID-NCBI, 1788) and DNMT3B genes (locusID-NCBI, 1789).

Article Snippet: Knock down of DNMT3A in HEK293 cells with DNMT3A-siRNA HEK-293 or stably transfected HEK293 cells were transfected with siRNA specific for DNMT3A (Santa Cruz, #sc-37757) for downregulation of endogenous DNMT3A gene expression.

Techniques:

Journal: TURKISH JOURNAL OF BIOLOGY

Article Title: The effects of DNA methyl transferases on antiaging klotho gene expression

doi: 10.3906/biy-1508-36

Figure Lengend Snippet: Figure 2. A. Western blot analysis of DNMT expression in HEK293 cells. The native DNMT3A and DNMT3B enzymes were visualized with rabbit polyclonal anti-DNMT3A and mouse monoclonal anti-DNMT3B antibodies, respectively. The DNMT enzymes tagged with the HA epitope were visualized with monoclonal anti-HA antibodies. The protein bands were visualized using SA-AP and the chromogenic substrate with BCIP-NBT. B. Expression and localization of DNMT enzymes in Swis3T3 cells. HA-tagged DNMT enzymes were visualized with monoclonal anti-HA antibody and FITC-conjugated monoclonal anti-mouse IgG antibodies. For visualization of native DNMT enzymes, rabbit polyclonal anti-DNMT3A, mouse monoclonal anti-DNMT3B, and FITC-conjugated secondary antibodies were used.

Article Snippet: Knock down of DNMT3A in HEK293 cells with DNMT3A-siRNA HEK-293 or stably transfected HEK293 cells were transfected with siRNA specific for DNMT3A (Santa Cruz, #sc-37757) for downregulation of endogenous DNMT3A gene expression.

Techniques: Western Blot, Expressing