Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) DOCK7 (TMW = 239 kDa, a ) and DNMT1 (TMW = 183 kDa, b ) protein interaction validated by DNMT1 co-immunoprecipitation and Western blot analysis by using a specific antibody against DOCK7 and DNMT1 in E14.5 cortical lysates. N = 3 experiments. + = DNMT1-antibody pulldown, - = IgG-antibody pulldown. ( c ) Representative images of N2a cells (grown for 48 h) co-stained with conjugated antibodies directed against DNMT1 (red) and DOCK7 (green), additionally stained with DAPI (blue), and captured by high-resolution STED microscopy. Scale bars: 5 µm. The white squares depict the magnification of the merge. Scale bars: 2 µm. Overlapping volumes are highlighted by white arrowheads. ( d, e ) Analysis of cytosolic colocalization between DNMT1 and DOCK7 by Pearson’s correlation coefficient (PCC) ( d ) and the percentage of colocalized volumes ( e ) in comparison to the corresponding rotated and randomized controls. n (ROIs) = 133; N (cells) = 45. ( f ) Predicted interaction between DNMT1 and DOCK7 using MD simulations and docking methods (left figure). Specific hot spot residues for the interaction between DNMT1 and DOCK7 are represented in the right figure. Hydrogen bonding and salt bridge interactions are shown as black dashes. ( g, h ) Inverted microphotographs of exemplary βIII-tubulin immunocytochemically stained cortical neurons (E14.5 + 2 DIV) transfected with control ( g ) or Dock7 ( h ) siRNA at 1 DIV for 24 h. Scale bars: 20 µm. ( i-l ) Analysis of morphological parameters, such as the length of the longest process ( i ), the number of processes ( j ), the branches per length summed across all processes likely representing dendrites ( k ), and the branches normalized to the longest process length likely representing axons ( l ). n (Ctrl siR) = 207 cells; n ( Dock7 siR) = 198 cells. N = 3 experiments. Two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Immunoprecipitation, Western Blot, Staining, Microscopy, Comparison, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: (a) Representative STED micrographs of a fixed N2a cell cultured for 48 h, labeled with MitoTracker™ Deep Red FM (Mito., false color green), DAPI (blue), and an antibody against DOCK7 (red). Scale bar: 5 µm. The white box indicates the magnified region shown in the merge (Scale bar: 2 µm). Nuclear signals were computationally removed to improve visualization of the cytosolic compartment. (b) Quantification of cytosolic colocalization between DOCK7 and mitochondria by Pearson’s correlation coefficient (PCC) compared to a rotated randomized control. n (ROIs) = 45; N (cells) = 15. Two-tailed Student’s t -test, p < 0.0001. (c) Representative images of cortical neurons (P0 + 4 DIV) co-transfected at DIV 3 with Alexa Fluor™ 555–labeled control siRNA (red) and MT-GFP plasmid (green) and imaged 24 h post-transfection. Scale bar: 10 µm. (d) Exemplary tracking of MT-GFP–labeled mitochondria in cortical neurons following control, Dnmt1 , or Dock7 siRNA treatment. Non-motile mitochondria are marked by white arrowheads, motile mitochondria by colored stars (each color indicates one mitochondrion). White boxes in overview images (first panel) mark the magnified regions shown over time. The last frame (sixth panel) depicts temporally color-coded mitochondrial trajectories. Scale bars: 5 µm. (e) Representative kymographs illustrating mitochondrial motility under the respective conditions. (f) Quantification of the ratio of non-motile to motile mitochondria in cortical neurons (P0 + 4 DIV) following control, Dnmt1 , or Dock7 siRNA-mediated knockdown for 24 h at DIV 3. N (Ctrl siR) = 35 ROIs; n ( Dnmt1 siR) = 39 ROIs; n ( Dock7 siR) = 33 ROIs. N = 3 independent experiments. Kruskal–Wallis test followed by Dunn’s post hoc multiple comparison test, p < 0.01, p < 0.001. Data are shown as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Cell Culture, Labeling, Control, Two Tailed Test, Transfection, Plasmid Preparation, Knockdown, Comparison

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) Live cell imaging analysis capturing mitochondria accumulation using the MitoTracker TM Deep Red FM prior to branch formation in N2a cells 24 h after control, Dnmt1 , or Dock7 siRNA transfection. The white squares in the first panel indicate the magnified regions shown for each time frame. (a) Inverted grayscale images of mitochondria accumulation at branch initiation sites, Scale bars: 10 µm. The MitoTracker TM integrated density, normalized to the corresponding integrated density of the same position in the first frame, at prospective branchpoints is shown in ( b ). n (Ctrl siR) = 22 cells with 54 events; n ( Dnmt1 siR) = 13 cells with 24 events; n ( Dock7 siR) = 21 cells with 43 events. N = 4 experiments. Two-way ANOVA followed by Tukey’s post-hoc multiple comparison test, ** p < 0.01. ( c, d ) Analysis of the branch formation time ( c ) and exemplary tracking of a branching event (from the first mitochondria accumulation puncta to the formation of the branch) ( d ). The white squares in the first panel indicate the magnified regions shown for each time frame. Scale bars: 20 µm. n (Ctrl siR) = 22 cells with 54 events; n ( Dnmt1 siR) = 13 cells with 24 events; n ( Dock7 siR) = 21 cells with 43 events. N = 4 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, ** p < 0.01, **** p < 0.0001. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Live Cell Imaging, Control, Transfection, Comparison

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) Microphotographs of cortical neurons ( a, Scale bars: 20 µm) and N2a cells ( b, Scale bars: 40 µm), which were transfected with control, Dnmt1 , or Dock7 siRNA for 24 h after growing for one day, co-stained for DAPI (blue), ßIII-tubulin (TUBB3, green), and acetylated tubulin (AcTUB, magenta). ( c, d ) Analysis of the integrated density for the acetylated tubulin (AcTUB) normalized to the βIII-tubulin (TUBB3) integrated density after the respective knockdown in cortical neurons ( c ) and N2a cells ( d ). N = 3 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, * p < 0.05, **** p < 0.0001. ( e, f ) Analysis of the acetylated tubulin (AcTUB) integrated density normalized to the βIII-tubulin (TUBB3) integrated density after the RG108 inhibitor treatment in cortical neurons ( e ) and N2a cells ( f ). N = 3 experiments. Two-tailed Student’s t-test, * p < 0.05. For ( c, e ): n (Ctrl siR) = 314 cells; n ( Dnmt1 siR) = 263 cells; n ( Dock7 siR) = 277 cells; n (DMSO) = 365 cells; n (RG108) = 358 cells. For ( d, f ): n (Ctrl siR) = 279 cells; n ( Dnmt1 siR) = 117 cells; n ( Dock7 siR) = 175 cells; n (DMSO) = 506 cells; n (RG108) = 525 cells. ( g, h ) Western blots revealing protein bands of STMN1 ( g , TMW = 17 kDa) and the phosphorylated version of STMN1 ( h , S16-P) (TMW = 17 kDa) in cortical single cell lysates (E14.5 + 2 DIV) treated previously with control, Dnmt1 , or Dock7 siRNA at 1 DIV for 24 h. γ-tubulin ( g , h , TUBG1, TMW = 51 kDa) was used as a housekeeper. ( i, j ) Analysis of the mean grey value of STMN1 normalized against TUBG1 ( i ) and the mean grey value of the phosphorylated version of STMN1 (S16-P) ( j ) normalized against STMN1. N = 3 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, * p < 0.05, ** p < 0.01. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Transfection, Control, Staining, Knockdown, Comparison, Two Tailed Test, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

doi: 10.1161/JAHA.121.022802

Figure Lengend Snippet: A , SGK3, phosphorylated (p‐) proteins of SGK3 Thr320 in neonatal cardiomyocytes transfected with Ad5:cTNT‐CON or Ad5:cTNT‐SGK3 were detected by Western blot. B through E , cardiomyocytes isolated from 100 P1 mice were transfected with Ad5:cTNT‐CON or Ad5:cTNT‐SGK3, and immunofluorescence staining was then used to evaluate the cardiomyocyte proliferation for EDU + (5639 cardiomyocytes in the Ad5:cTNT‐SGK3 group and 5996 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( B ); Ki67 + (6511 cardiomyocytes in the Ad5:cTNT‐SGK3 group and 6852 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( C ); pH3 + (8391 cardiomyocytes in the Ad5:SGK3 group and 7121 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( D ); and Aurora B + (8503 cardiomyocytes in the Ad5:SGK3 group and 14 162 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( E ). F , SGK3 expression in neonatal cardiomyocytes transfected with Ad5:cTNT‐SGK3i or Ad5:cTNT‐CONi were analyzed by Western blot analysis. G through I , Immunofluorescence staining of cardiomyocytes isolated from 100 P1 mice transfected with Ad5:cTNT‐SGK3i or Ad5:cTNT‐CONi and quantification of EDU + (4716 cardiomyocytes in the Ad5:SGK3i group and 4736 cardiomyocytes in the Ad5:cTNT‐CONi group, n=6 ( G ); Ki67 + (6388 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 9583 cardiomyocytes in the Ad5:cTNT‐CONi group, n=6 ( H ); and pH3 + (5642 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 6576 cardiomyocytes in the Ad5:cTNT‐CONi group, n=6 ( I ). J and K , Cell flow cytometry was performed to detect the cell cycle of cardiomyocytes after SGK3 overexpression or inhibition. L , Terminal deoxynucleotidyl transferase‐mediated dUTP in situ nick end labeling (TUNEL) staining was performed to evaluate the effect of Ad5:cTNT‐SGK3 on oxygen glucose deprivation/reoxygenation (OGD/R)‐induced neonatal mouse cardiomyocyte apoptosis (3657 cardiomyocytes in the Ad5:cTNT‐CON group, 3017 cardiomyocytes in the Ad5:cTNT‐SGK3 group, 2049 cardiomyocytes in the OGD/R+Ad5:cTNT‐CONi group, and 1303 cardiomyocytes in the OGD/R+Ad5:cTNT‐SGK3 group, n=6). The cells indicated by the arrows are immunofluorescence‐positive cardiomyocytes. Data are presented as mean±SEM. * P ≤0.05; ** P ≤0.01; *** P ≤0.001. Ad5:cTNT‐CON indicates control adenovirus serotype 5; Ad5:cTNT‐CONi, knockdown control adenovirus serotype 5; Ad5:cTNT‐SGK3, cardiomyocyte‐specific SGK3 overexpression adenovirus serotype 5; Ad5:cTNT‐SGK3i, cardiomyocyte‐specific SGK3 knockdown adenovirus serotype 5; CON, control; CONi, knockdown control; cTNT, cardiac troponin T; EDU, 5‐ethynyl‐2′‐deoxyuridine; G1, G1 phase; G2/M, G2/M phase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; ki67+, ki67 positive; NS, no significance; P1, postpartum day 1; ph3+:phospho‐histone H3 positive; S, S phase; and SGK3i, serine/threonine‐protein kinase 3 knockdown.

Article Snippet: To identify cell‐cycle activities and cytokinesis, the Click‐iT EdU Imaging Kits (Thermo Fisher), anti‐Ki67 antibody (Abcam; ab16667), anti‐Aurora B antibody (Abcam; ab2254), and anti‐phosphorylated‐histone 3 (pH3) antibodies (CST; 9701) were used to culture sections and formaldehyde‐fixed cardiomyocytes.

Techniques: Transfection, Western Blot, Isolation, Immunofluorescence, Staining, Expressing, Flow Cytometry, Over Expression, Inhibition, In Situ, End Labeling, TUNEL Assay, Control, Knockdown

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

doi: 10.1161/JAHA.121.022802

Figure Lengend Snippet: A , Experimental pattern: myocardium targeting SGK3 overexpression of adeno‐associated virus serotype 9 (AAV9:cTNT‐SGK3) or control (AAV9:cTNT‐CON) was intraperitoneally injected into P1 mice, and intraperitoneally injected EDU solution at P8, P10, and P12, and the hearts were then collected at P14 for relevant experiments. B through E , After 14 days treatment of AAV9:cTNT‐SGK3 or AAV9:cTNT‐CON in P1 mice, immunofluorescence staining was then performed at P14 to evaluate the cardiomyocyte proliferation for EDU + (12 565 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 12 678 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( B ); pH3 + (10 067 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 5788 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( C ); Ki67 + (9908 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 10 241 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( D ); and Aurora B + (18 132 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 17 707 cardiomyocytes in the AAV9:cTNT‐CON group, n=6 ( E ). F , Terminal deoxynucleotidyl transferase‐mediated dUTP in situ nick end labeling (TUNEL) staining was used to evaluate the effect of AAV9:cTNT‐SGK3 or AAV9:CTNT‐CON on cardiomyocyte apoptosis in mice at P14 (5723 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 5457 cardiomyocytes in the AAV9:cTNT‐CON group, n=6). G , Heart weight/body weight (HW/BW) ratio and cardiac morphology between AAV9:cTNT‐SGK3 (n=5) and AAV9:cTNT‐CON mice (n=5) at P14. H , Survival rate was analyzed by between AAV9:cTNT‐CON (n=10) and AAV9:cTNT‐SGK3 (n=10). I , Wheat germ agglutinin (WGA) immunofluorescence was used to detect the cardiomyocyte size between AAV9:cTNT‐SGK3 and AAV9:cTNT‐CON mice at P14 (14 687 cardiomyocytes in the AAV9:cTNT‐SGK3 group and 14 582 cardiomyocytes in the AAV9:cTNT‐CON group, n=6). Data are presented as mean±SEM. *** P ≤0.001. CON indicates control; cTNT, cardiac troponin T; EDU, 5‐ethynyl‐2′‐deoxyuridine; i.p., intraperitoneal; ki67+, ki67 positive; NS, no significance; P1, postpartum day 1; P14, postnatal day 14; and pH3+, phospho‐histone H3 positive.

Article Snippet: To identify cell‐cycle activities and cytokinesis, the Click‐iT EdU Imaging Kits (Thermo Fisher), anti‐Ki67 antibody (Abcam; ab16667), anti‐Aurora B antibody (Abcam; ab2254), and anti‐phosphorylated‐histone 3 (pH3) antibodies (CST; 9701) were used to culture sections and formaldehyde‐fixed cardiomyocytes.

Techniques: Over Expression, Virus, Control, Injection, Immunofluorescence, Staining, In Situ, End Labeling, TUNEL Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

doi: 10.1161/JAHA.121.022802

Figure Lengend Snippet: A , Experimental pattern: adenovirus serotype 5 SGK3i (Ad5:cTNT‐SGK3i) or control (Ad5:cTNT‐CONi) was injected into border myocardium after apical resection (AR) in P1 mice using microinjector. EDU solution was then intraperitoneally injected at 4 days post resection (dpr). Finally, the cardiomyocyte proliferation was detected at 6 dpr, and cardiac function was evaluated at 22 dpr. B and C , Cardiac function of ejection fraction and fractional shortening between Ad5:cTNT‐SGK3i or Ad5:cTNT‐CONi treated mice at 1 and 22 days after AR (Ad5:cTNT‐SGK3i group, n=13; Ad5:cTNT‐CONi, n=13). D through F , Immunofluorescence staining was used to evaluate the cardiomyocyte proliferation between Ad5:cTNT‐SGK3i– or Ad5:cTNT‐CONi–treated mice at P6 for EDU + (11 873 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 11 755 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( B ); pH3 + (15 210 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 15 084 cardiomyocytes in the Ad5:cTNT‐CON group, n=6( C ); and Ki67 + (12 290 cardiomyocytes in the Ad5:cTNT‐SGK3i group and 13 119 cardiomyocytes in the Ad5:cTNT‐CON group, n=6 ( D ). G , Masson staining of mouse ventricular cross‐sections between Ad5:cTNT‐SGK3i– or Ad5:cTNT‐CONi–treated mice at 22 days after AR (n=7 in each group). Data are presented as mean±SEM. * P ≤0.05; ** P ≤0.01; *** P ≤0.001. CON indicates control; CONi, knockdown control; cTNT, cardiac troponin T; EDU, 5‐ethynyl‐2′‐deoxyuridine; ki67+, ki67 positive; NS, no significance; P1, postpartum day 1; and pH3+, phospho‐histone H3 positive.

Article Snippet: To identify cell‐cycle activities and cytokinesis, the Click‐iT EdU Imaging Kits (Thermo Fisher), anti‐Ki67 antibody (Abcam; ab16667), anti‐Aurora B antibody (Abcam; ab2254), and anti‐phosphorylated‐histone 3 (pH3) antibodies (CST; 9701) were used to culture sections and formaldehyde‐fixed cardiomyocytes.

Techniques: Control, Injection, Immunofluorescence, Staining, Knockdown