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: 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 ) Exemplary brain sections (300 µm) from P5 brains of pups, which were intrauterine electroporated at E12.5 with either CRISPRi-Ctrl-GFP ( a ) or CRISPRi-Dnmt1-GFP ( b ), followed by immunostaining for GFP (CRISPRi-Ctrl-GFP/CRISPRi-Dnmt1-GFP, green), CTIP2 (red), and DAPI (blue). Distinct cortical layers are labeled from I to VI. Scale bars: 200 µm. ( c, d ) Exemplary images (left) and 3D-reconstructions (right) of exemplary GFP-positive laver V cortical neurons from the CRISPRi-Ctrl-GFP ( c ) or the CRISPRi-Dnmt1-GFP ( d ) conditions at P5. The white/black arrow heads highlight basal dendrites. Scale bars: 10 µm. ( e ) Analysis of the absolute basal dendrite number. n (CRISPRi-Ctrl-GFP) = 25 cells; n (CRISPRi-Dnmt1-GFP) = 25 cells. N = 4 experiments. Welch’s t-test, * p < 0.05. ( f, g ) Inverted microphotographs of exemplary βIII-tubulin immunocytochemically stained cortical neurons (E14.5 + 2 DIV) previously transfected with control ( f ) or Dnmt1 ( g ) siRNA at 1 DIV for 24 h. Scale bars: 20 µm. ( h-k ) Quantitative analysis of the length of the longest process ( h ), the number of processes ( i ), the branches normalized to the sum of the lengths of all processes (except the axon) presumably representing dendrites ( j ), and the branches per longest process length likely representing the axon ( k ). n (Ctrl siR) = 207 cells; n ( Dnmt1 siR) = 197 cells. N = 3 experiments. Two-tailed Student’s t-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: Immunostaining, Labeling, Staining, 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 ) Scheme of the different generated Dnmt1 expression constructs demonstrating all their relevant functional domains, tags, and promotor. ( b ) Representative fluorescence microphotographs of N2a cells expressing siRNA resistant mNG-DNMT1 (WT, ΔCat, ΔpCat, ΔNLS) plasmids (green) 24 h post co-transfection with Dnmt1 siRNA and followed by immunostaining with DAPI (blue). Scale bars: 10 µm. ( c ) Representative microphotograph of cortical neurons (E14.5 + 2 DIV) co-transfected with Dnmt1 siRNA and the different Dnmt1 plasmids (green) at 1 DIV for 24 h, which were later immunocytochemically stained for βIII-tubulin (TUBB3, magenta) and DAPI (blue). The white squares represent the respective magnification of the merge. Scale bars: 20 µm. ( d ) Exemplary inverted microphotographs of βIII-tubulin-stained cortical neurons (E14.5 + 2 DIV) under the same co-transfection conditions. Scale bars: 20 µm. ( e, f ) Analysis of the branches normalized to the longest process length presumably representing axons ( f ) and branches per length cumulated across all remaining process lengths likely representing dendrites ( e ). “+” indicates neurons, which were also successfully co-transfected with Dnmt1 siRNA for the downregulation of the endogenous Dnmt1 expression in addition to the Dnmt1 plasmids. n (D1-WT) = 141 cells; (D1-ΔCat) = 136 cells; n (D1-ΔpCat) = 98 cells; n (D1-ΔNLS) = 150 cells; n (Ctrl siR) = 153 cells; n ( Dnmt1 siR) = 146 cells. N = 3 experiments. One-way ANOVA with Tukey’s post-hoc multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** 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: Generated, Expressing, Construct, Functional Assay, Fluorescence, Cotransfection, Immunostaining, Transfection, Staining, 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) Immunocytochemical staining for DNMT1 (red) and DAPI (blue) in a cortical neuron (E14.5 + 2 DIV) (a) and an N2a cell (b) , visualized by high-resolution STED microscopy. Scale bars: 5 µm. (c, d) Volcano plots showing proteins co-immunoprecipitated with DNMT1 from murine brain tissue lysates (3.5 months; c, Supplementary Data 1) and N2a cell lysates (d, Supplementary Data 2), identified by mass spectrometry. N = 4 biological replicates for each condition. (e, f) Gene Ontology (GO) enrichment analysis of DNMT1 co-immunoprecipitated proteins from murine brain tissue (e) and N2a cells (f) , performed using the STRING database and ShinyGO (v0.82). (g) Representative STED micrograph of a fixed N2a cell cultured for 48 h, labeled with MitoTracker™ Deep Red FM (shown in false color green), DAPI (blue), and an antibody against DNMT1 (red). Scale bar: 5 µm. The white box indicates the magnified region shown in the inset (Scale bar: 2 µm). Nuclear signals were computationally removed to enhance visualization of the cytosolic compartment for further analysis. (h) Quantification of cytosolic colocalization between DNMT1 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 < 0.05, p < 0.01, p < 0.001, p < 0.0001.

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: Staining, Microscopy, Immunoprecipitation, Mass Spectrometry, Cell Culture, Labeling, 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 images of cortical neurons (P0 + 4 DIV) co-transfected at DIV 3 with MT-dsRed (red) and the indicated Dnmt1 plasmid constructs (green) for 24 h. Scale bars: 10 µm. (b) Exemplary tracks of MT-dsRed–labeled mitochondria in cortical neurons under the respective Dnmt1 expression conditions. Non-motile mitochondria are indicated by white arrowheads; motile mitochondria are marked with colored stars (each color corresponds to one mitochondrion). White boxes in the overview images (first panel) indicate magnified regions shown across time frames. The final frame (sixth panel) displays temporal color coding of mitochondrial trajectories. Scale bars: 5 µm. (c) Representative kymographs illustrating mitochondrial motility under the respective conditions. (d) Quantification of the ratio of non-motile to motile mitochondria in cortical neurons (P0 + 4 DIV). “+” indicates neurons co-transfected with Dnmt1 siRNA to downregulate endogenous Dnmt1 expression in addition to the respective Dnmt1 expression constructs. N (D1-WT) = 39 ROIs; n (D1-ΔpCat) = 36 ROIs; n (D1-ΔNLS) = 35 ROIs; n (Ctrl siR) = 71 ROIs; n ( Dnmt1 siR) = 75 ROIs. N = 4-7 independent experiments. Kruskal–Wallis test followed by Dunn’s post hoc multiple comparison test, p < 0.01, p < 0.001. Data represent 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, Plasmid Preparation, Construct, Labeling, Expressing, 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 ) 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: Genes & Diseases

Article Title: RAB26 promotes prostate cancer progression via the MAPK/ERK-TWIST1 signaling axis

doi: 10.1016/j.gendis.2025.101689

Figure Lengend Snippet: RAB26 promotes epithelial–mesenchymal transition and stem cell-like characteristics of prostate cancer cells via MAPK-ERK/TWIST1 axis. (A) Western blotting detected MAPK/ERK1/2 signaling pathway-related proteins in PC3, LNCaP, and 22RV1 cells. (B) After serum starvation, western blotting detected epidermal growth factor (EGF)-treated cells to determine the phosphorylation of ERK1/2 at a specified time point after serum starvation. (C) Immunofluorescence assay detected the binding condition of RAB26 (red) and GM130 (yellow). (D) Western blotting detected ERK1/2 and p-ERK1/2 in PC3 after brefeldin A (BFA) treatment. (E) Immunofluorescence assay detected the binding condition of RAB26 (red) and p-ERK1/2 (yellow). (F) Western blotting detected nucleocytoplasmic distribution of p-ERK1/2 and RAB26. (G) Western blotting detected nucleocytoplasmic distribution of p-ERK1/2, TWIST1, and RAB26. (H – J) Immunofluorescence assay detected RAB26/p-ERK1/2 promoting TWIST1 nuclear translocation in PC3, LNCaP, and 22RV1 cells. (K) Combined JASPAR and TCGA predicted transcription factors bound by RAB26 promoter. (M, N) A double luciferase gene reporting experiment detected luciferase activity. (O, P) Quantitative reverse-transcription PCR detected gene expression.

Article Snippet: The confocal immunofluorescence staining steps were the same as immunofluorescence staining, but the fluorescence labeling was performed using the FlexAble CoraLite® Plus 555 Antibody Labeling Kit (KFA002, Proteintech, China) for the RAB26 and TWIST1 and the FlexAble CoraLite® Plus 647 Antibody Labeling Kit for Rabbit IgG (KFA003, Proteintech, China) for the p-ERK and GM130 as per the manufacturer's protocol.

Techniques: Western Blot, Phospho-proteomics, Immunofluorescence, Binding Assay, Translocation Assay, Luciferase, Activity Assay, Reverse Transcription, Gene Expression

Journal: Genes & Diseases

Article Title: RAB26 promotes prostate cancer progression via the MAPK/ERK-TWIST1 signaling axis

doi: 10.1016/j.gendis.2025.101689

Figure Lengend Snippet: Analysis of clinical correlation between RAB26 expression in prostate cancer. (A) Expression density map of single-cell sequencing cell typing in prostate cancer. (B) UMAP diagram of RAB26 in prostate cancer cells. (C) Expression of RAB26 in different cell types. (D) Expression of RAB26 in different tumors. (E) RAB26 and progression-free survival. (F) RAB26 and disease-free survival. (G) The relationship between RAB26 and the T stage of prostate cancer. (H) The relationship between RAB26 and lymph node metastasis in prostate cancer. (I) The relationship between RAB26 and different Gleason scores. (J) Differential expression of RAB26 between normal prostate tissue and prostate cancer tissue. (K) Western blotting detected RAB26 in the tumor and para-carcinoma tissues of patients with prostate cancer. (L) The immunohistochemical staining of RAB26 in patients with prostate cancer. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The confocal immunofluorescence staining steps were the same as immunofluorescence staining, but the fluorescence labeling was performed using the FlexAble CoraLite® Plus 555 Antibody Labeling Kit (KFA002, Proteintech, China) for the RAB26 and TWIST1 and the FlexAble CoraLite® Plus 647 Antibody Labeling Kit for Rabbit IgG (KFA003, Proteintech, China) for the p-ERK and GM130 as per the manufacturer's protocol.

Techniques: Expressing, Sequencing, Quantitative Proteomics, Western Blot, Immunohistochemical staining, Staining

Journal: Genes & Diseases

Article Title: RAB26 promotes prostate cancer progression via the MAPK/ERK-TWIST1 signaling axis

doi: 10.1016/j.gendis.2025.101689

Figure Lengend Snippet: The function of RAB26 was confirmed by in vitro experiments. (A, B) Quantitative reverse-transcription PCR and western blotting detected the expression of RAB26 in different cell lines. (C) RAB26 was successfully overexpressed in PC3 cells and knocked down in LNCaP and 22RV1 cells. (D) The effect of RAB26 on the proliferation of PC3, LNCaP, and 22RV1 cells was evaluated using CCK-8 assay. (E) The effect of RAB26 on the ability of colony formation in PC3, LNCaP, and 22RV1 cells. (F) The effect of RAB26 on the proliferation of PC3, LNCaP, and 22RV1 cells was evaluated using EdU. (G, H) The effect of RAB26 on apoptosis and the cell cycle was detected by flow cytometry. (I) The effect of RAB26 on the migration and invasion of PC3, LNCaP, and 22RV1 cells was detected by transwell assay. (J) The effect of RAB26 on the migration of Caki-1 cells was detected by scratch test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The confocal immunofluorescence staining steps were the same as immunofluorescence staining, but the fluorescence labeling was performed using the FlexAble CoraLite® Plus 555 Antibody Labeling Kit (KFA002, Proteintech, China) for the RAB26 and TWIST1 and the FlexAble CoraLite® Plus 647 Antibody Labeling Kit for Rabbit IgG (KFA003, Proteintech, China) for the p-ERK and GM130 as per the manufacturer's protocol.

Techniques: In Vitro, Reverse Transcription, Western Blot, Expressing, CCK-8 Assay, Flow Cytometry, Migration, Transwell Assay

Journal: Genes & Diseases

Article Title: RAB26 promotes prostate cancer progression via the MAPK/ERK-TWIST1 signaling axis

doi: 10.1016/j.gendis.2025.101689

Figure Lengend Snippet: RAB26 promotes epithelial–mesenchymal transition and stem cell-like characteristics of prostate cancer cells. (A, B) The results of GSEA enrichment analysis and representative GSEA data. (C, D) Western blotting and quantitative reverse-transcription PCR detected the expression of epithelial-mesenchymal transition-related marker proteins in PC3, LNCaP, and 22RV1 cells. (E) The relationship between RAB26 and mRNA score. (F) Quantitative reverse-transcription PCR detected the expression of stem cell-related markers in PC3, LNCaP, and 22RV1 cells. (G) RAB26 affected the formation of tumor stem cell spheres in PC3, LNCaP, and 22RV1 cells. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The confocal immunofluorescence staining steps were the same as immunofluorescence staining, but the fluorescence labeling was performed using the FlexAble CoraLite® Plus 555 Antibody Labeling Kit (KFA002, Proteintech, China) for the RAB26 and TWIST1 and the FlexAble CoraLite® Plus 647 Antibody Labeling Kit for Rabbit IgG (KFA003, Proteintech, China) for the p-ERK and GM130 as per the manufacturer's protocol.

Techniques: Western Blot, Reverse Transcription, Expressing, Marker

Journal: Genes & Diseases

Article Title: RAB26 promotes prostate cancer progression via the MAPK/ERK-TWIST1 signaling axis

doi: 10.1016/j.gendis.2025.101689

Figure Lengend Snippet: RAB26 promotes epithelial–mesenchymal transition and stem cell-like characteristics of prostate cancer cells via MAPK-ERK/TWIST1 axis. (A) Western blotting detected MAPK/ERK1/2 signaling pathway-related proteins in PC3, LNCaP, and 22RV1 cells. (B) After serum starvation, western blotting detected epidermal growth factor (EGF)-treated cells to determine the phosphorylation of ERK1/2 at a specified time point after serum starvation. (C) Immunofluorescence assay detected the binding condition of RAB26 (red) and GM130 (yellow). (D) Western blotting detected ERK1/2 and p-ERK1/2 in PC3 after brefeldin A (BFA) treatment. (E) Immunofluorescence assay detected the binding condition of RAB26 (red) and p-ERK1/2 (yellow). (F) Western blotting detected nucleocytoplasmic distribution of p-ERK1/2 and RAB26. (G) Western blotting detected nucleocytoplasmic distribution of p-ERK1/2, TWIST1, and RAB26. (H – J) Immunofluorescence assay detected RAB26/p-ERK1/2 promoting TWIST1 nuclear translocation in PC3, LNCaP, and 22RV1 cells. (K) Combined JASPAR and TCGA predicted transcription factors bound by RAB26 promoter. (M, N) A double luciferase gene reporting experiment detected luciferase activity. (O, P) Quantitative reverse-transcription PCR detected gene expression.

Article Snippet: The confocal immunofluorescence staining steps were the same as immunofluorescence staining, but the fluorescence labeling was performed using the FlexAble CoraLite® Plus 555 Antibody Labeling Kit (KFA002, Proteintech, China) for the RAB26 and TWIST1 and the FlexAble CoraLite® Plus 647 Antibody Labeling Kit for Rabbit IgG (KFA003, Proteintech, China) for the p-ERK and GM130 as per the manufacturer's protocol.

Techniques: Western Blot, Phospho-proteomics, Immunofluorescence, Binding Assay, Translocation Assay, Luciferase, Activity Assay, Reverse Transcription, Gene Expression

Journal: Genes & Diseases

Article Title: RAB26 promotes prostate cancer progression via the MAPK/ERK-TWIST1 signaling axis

doi: 10.1016/j.gendis.2025.101689

Figure Lengend Snippet: RAB26 promotes prostate cancer proliferation and metastasis in vivo . (A) Gross view of mice and tumors. (B) Growth curve of tumor volume. (C) The weight of the tumor. (D) Representative hematoxylin-eosin staining images in the xenograft model of prostate cancer. (E) Western blotting verified the expression of RAB26 in tumor tissue. (F) Representative immunohistochemistry images of epithelial-mesenchymal transition-related markers in tumor tissue. (G) Representative immunohistochemistry images of Bmil, NANOG, and SOX2 in tumor tissue. (H) Imaging of living small animals and gross images of lung metastases. (I) Representative hematoxylin-eosin staining images of a lung metastatic tumor. (J) Representative immunohistochemistry images of MMP2 and MMP9 in lung metastatic tumor. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The confocal immunofluorescence staining steps were the same as immunofluorescence staining, but the fluorescence labeling was performed using the FlexAble CoraLite® Plus 555 Antibody Labeling Kit (KFA002, Proteintech, China) for the RAB26 and TWIST1 and the FlexAble CoraLite® Plus 647 Antibody Labeling Kit for Rabbit IgG (KFA003, Proteintech, China) for the p-ERK and GM130 as per the manufacturer's protocol.

Techniques: In Vivo, Staining, Western Blot, Expressing, Immunohistochemistry, Imaging