Journal: PLOS One

Article Title: Targeted knockdown of DNA methyltransferase 3a (DNMT3a) unlocks dedifferentiation and neurogenic potential in mouse retinal Müller glia

doi: 10.1371/journal.pone.0337891

Figure Lengend Snippet: (A) Experimental design for early-neuron marker βIII-tubulin detection, time course for sample acquisition and chemical treatment. (B) Representative immunofluorescences of MG at 48 h after treatments, cultured in basal growth media, CTRL: control, NMDA: exposure to NMDA 100 µM, MOCK: electroporation without plasmid, dCas9: transfection of empty plasmid, DNMT3a KD: CRISPR interference-mediated Dnmt3a knockdown, DNMT3a KD NMDA: Dnmt3a knockdown with simultaneous exposure to NMDA 100 µM; show GS (red), βIII-tubulin (green), and nuclei (DAPI, blue). (C) Percentage of positive cells to GS and βIII-tubulin, where all conditions present constant expression of GS, while βIII-tubulin percentage of positive cells were increased in DNMT3a KD and DNMT3a KD NMDA. (D) Quantification of fluorescence intensity of GS marks at 48 h after treatment, no significant differences were observed across conditions. (E) Quantification of fluorescence intensity of βIII-tubulin shows an increase in DNMT3a KD and DNMT3a KD NMDA groups, consistent with the increase in βIII-tubulin percentage of positive cells. Scale bars = 20 µm, 40X. Percentage of positive cells for each marker were presented as bar graphic for mean ± SEM from three independent experiments, counting a minimum of 10 non-overlapped fields and at least 150 cells per group. *p < 0.05, **p < 0.01, ns = no significant (one-way ANOVA with Tukey post hoc test).

Article Snippet: After that the samples were incubated with following primary antibodies: mouse monoclonal anti-βlll tubulin antibody (Santa Cruz sc-80005) (1:300); rabbit polyclonal anti-GS antibody (Abcam ab73593) (1:300); mouse monoclonal anti-nestin antibody (Santa Cruz sc-23927) (1:200); rabbit polyclonal anti-Lin28 (Abcam ab46020) (1:200) diluted in 1:3 blocking solution at 4°C overnight.

Techniques: Marker, Cell Culture, Control, Electroporation, Plasmid Preparation, Transfection, CRISPR, Knockdown, Expressing, Fluorescence

Journal: PLOS One

Article Title: Targeted knockdown of DNA methyltransferase 3a (DNMT3a) unlocks dedifferentiation and neurogenic potential in mouse retinal Müller glia

doi: 10.1371/journal.pone.0337891

Figure Lengend Snippet: (A) Experimental design for early-neuron marker βIII-tubulin induction with GABA after CRISPRi transfection, showing the time course followed for sample acquisition and chemical treatment. (B) Representative images for immunofluorescence of MG cultured 48 h in growth medium and then cultured for 9 days in differentiation medium with GABA 100 µM. Images show GS (red), βIII-tubulin (green), and nuclei (DAPI, blue). Control conditions (CTRL, MOCK, dCas9) maintain GS expression with negligible βIII-tubulin signal. Meanwhile in NMDA, DNMT3a KD and DNMT3a KD NMDA βIII-tubulin positive cells were present, and GS expression was maintained. CTRL: control, NMDA: exposure to NMDA 100 µM, MOCK: electroporation without plasmid, dCas9: transfection of empty plasmid, DNMT3a KD: CRISPR interference-mediated Dnmt3a knockdown, DNMT3a KD NMDA: Dnmt3a knockdown with simultaneous exposure to NMDA 100 µM. Scale bars = 20 µm, 40X. (C) Quantification of percentage of positive cells to each marker, all conditions present constant expression of GS, while βIII-tubulin percentage of positive cells were increased in NMDA, DNMT3a KD and DNMT3a KD NMDA. (D) Quantification of fluorescence intensity of GS marks at 48 h after treatment, no significant differences were observed across conditions. (E) Quantification of fluorescence intensity of βIII-tubulin shows an increase in NMDA, DNMT3a KD and DNMT3a KD NMDA groups. Data were presented as violin plots of at least three independent experiments, counting a minimum of 10 non-overlapped fields and at least 150 cells per group. *p < 0.05, ***p < 0.001, ns = no significant (one-way ANOVA with Tukey’s post hoc test).

Article Snippet: After that the samples were incubated with following primary antibodies: mouse monoclonal anti-βlll tubulin antibody (Santa Cruz sc-80005) (1:300); rabbit polyclonal anti-GS antibody (Abcam ab73593) (1:300); mouse monoclonal anti-nestin antibody (Santa Cruz sc-23927) (1:200); rabbit polyclonal anti-Lin28 (Abcam ab46020) (1:200) diluted in 1:3 blocking solution at 4°C overnight.

Techniques: Marker, Transfection, Immunofluorescence, Cell Culture, Control, Expressing, Electroporation, Plasmid Preparation, CRISPR, Knockdown, Fluorescence

Journal: PLOS One

Article Title: Targeted knockdown of DNA methyltransferase 3a (DNMT3a) unlocks dedifferentiation and neurogenic potential in mouse retinal Müller glia

doi: 10.1371/journal.pone.0337891

Figure Lengend Snippet: Representative immunofluorescences of MG at 48 h and 9 days after CRISPRi transfection with simultaneous addition of NMDA 100 µM, where we could observe coexpresión of glial (GS, red) and early-neuronal (βIII-tubulin, green) markers indicating a partial cell reprogramming. DNMT3a KD NMDA: Dnmt3a knockdown with simultaneous exposure to NMDA 100 µM; show GS (red), βIII-tubulin (green), and nuclei (DAPI, blue). Scale bars = 50 µm, 40X.

Article Snippet: After that the samples were incubated with following primary antibodies: mouse monoclonal anti-βlll tubulin antibody (Santa Cruz sc-80005) (1:300); rabbit polyclonal anti-GS antibody (Abcam ab73593) (1:300); mouse monoclonal anti-nestin antibody (Santa Cruz sc-23927) (1:200); rabbit polyclonal anti-Lin28 (Abcam ab46020) (1:200) diluted in 1:3 blocking solution at 4°C overnight.

Techniques: Transfection, Knockdown