Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: (A) Schematic of the Dock7 wild type (+) and Dock7 exons 3–4 deletion ( Dock7-em2) transgenic allele. Image created with BioRender.com. (B) Predicted translation of the Dock7+ and Dock7-em2 alleles. Image created with BioRender.com. (C) Representative images of Dock7 +/+ , Dock7 +/em2 , and Dock7 em2/em2 mice.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: (A) RNA expression of Dock7 exons 3–4, exons 11–12, and exons 34–35 was analyzed in BMSCs differentiated for 7 days in osteogenic media. Points represent individual replicates for all 3 experiments. (B) Quantitative protein analysis of DOCK7 peptides was performed by mass spectrometry in BMSC isolated from Dock7 +/+ and Dock7 em2/em2 mice. N=2 mice/group. Points represent replicates from all mice. (C) DOCK7 peptides were detected by mass spectrometry and used to quantify total DOCK7 levels. High confidence (green), medium confidence (yellow), and low confidence (red) DOCK7 peptides are indicated. The protein sequence corresponding to DOCK7 exons 3–4, the DHR1 domain, and the DHR2 domain is underlined and labeled.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: RNA Expression, Mass Spectrometry, Isolation, Sequencing, Labeling

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet:

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques:

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: Body composition was measured by DXA analysis in male and female Dock7 em2/em2 mice and compared to Dock7 +/+ control mice. Data was analyzed by 2-way ANOVA and Holm-Šídák’s test for post-hoc analysis. N=14–15 mice/group. Points represent each mouse.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: Control

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: Osteogenic differentiation was assessed in BMSCs that were isolated from Dock7 +/+ and Dock7 em2/em2 mice after 7 days of osteogenic differentiation. (A) Representative image of von Kossa and alkaline phosphatase staining. (B) Expression of the osteoblast-related genes Runx2 , AlpI , and Bglap . Points represent individual replicates for all 3 experiments.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: Isolation, Staining, Expressing

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: Research

Article Title: CCN5 Drives Leydig Cell Aging and Testicular Dysfunction: Insights into Fibrosis, Lipid Dysregulation, and Therapeutic Potential

doi: 10.34133/research.0762

Figure Lengend Snippet: CCN5 promotes LCs senescence by down-regulating RNF213. (A) This table shows the top 10 proteins that combined with CCN5 identified by LC-MS/MS. (B) Bar plot shows the enrichment analysis of proteins that combined with CCN5. (C) The LCs (top panel) and 293t (bottom panel) cells lysates were immunoprecipitated by anti-FLAG antibody (fused with CCN5) or IgG with protein A/G magnetic beads. The precipitated proteins were analyzed by Western blot using anti-DOCK7 and anti-RNF213 antibody, respectively. (D) Immunofluorescence co-staining of RNF213 (red) and CCN5 (green) in young and old mice testis. The scale bar represents 100 μm. n = 10 different regions. (E) Scatter plot showing the correlation between CCN5 and RNF213 expression levels in the testicular interstitial region. (F) Western blot shows the level of RNF213 in LCs after recombinant CCN5 protein with CCN5(ΔSP) or CCN5(wt) overexpression. n = 3 technical repeats. (G) Western blot analysis to validate the knockdown efficiency of RNF213 in LCs. n = 3 technical repeats. (H) Western blot shows the fold change of FOXO1, FOXO3, p16, and p21 in LCs after knockdown of RNF213. n = 3 technical repeats. (I) ELISA was used to measure the change of testosterone concentration in the culture supernatant of LCs after knockdown of RNF213. n = 3 technical repeats. (J) SA-β-gal staining of LCs with RNF213 knockdown. The scale bar represents 20 μm. n = 5 technical repeats. (K) Crystal violet staining marks colony formation in LCs with RNF213 knockdown. n = 2 technical repeats. (L) Oil red staining of LCs with RNF213 knockdown. The scale bar represents 10 μm. n = 10 cells.

Article Snippet: The following primary antibodies were used for immunoblotting: DYKDDDDK tag Monoclonal antibody (Proteintech, 66008-4-IG, 1:5,000), RNF213 Antibody (Novus, NBP1-88466, 1:1,000), Smad2 (D43B4) XP Rabbit mAb (CST, 5339T, 1:1,000), Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb (CST, 4511T, 1:1,000), Smad3 Rabbit pAb (Abclonal, A11471, 1:1,000), Phospho-Smad3-S423/S425 Rabbit mAb (Abclonal, AP0727, 1:1,000), alpha-SMA Antibody (Affinity Biosciences, AF1032, 1:1,000), ccn5-rabbit-polyclonal-antibody (Origene, TA383325; 1:1,000), and DOCK7 Polyclonal antibody (proteintech, 13000-1-AP, 1:1,000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Magnetic Beads, Western Blot, Immunofluorescence, Staining, Expressing, Recombinant, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cardiovascular Diabetology

Article Title: ICAM1 blockade improves ischemic muscle reperfusion in diabetic mice

doi: 10.1186/s12933-025-02573-3

Figure Lengend Snippet: Blood flow recovery in the ischemic foot of diabetic mice is impaired from day 14 following HLI surgery. A – B Diabetes was induced, or not, in 8 week-old C57BL/6 mice via STZ injections, 12 weeks later mice underwent HLI surgery. C – D Lepr db/db male mice and their control littermates (Lepr db /+) underwent HLI surgery at 12 weeks of age. E – F 8 week-old C57BL/6 mice were fed with HFD or a normal chow diet; one month later HFD-fed mice received STZ administration to induce hyperglycemia. At 20 weeks of age, mice underwent HLI surgery. Ischemic foot perfusion was assessed via laser Doppler perfusion imaging 7, 14, and 28 days after surgery. A , C , E Representative images taken 28 days after surgery are shown. B , D , F Ischemic foot perfusion was quantified as the perfusion ratio of ischemic foot over control foot ( n = 10 mice in each group). G – H The number of necrotic toes was counted ( n = 10 mice in each group). Mann Whitney tests

Article Snippet: C57BL/6J, and Lepr db mice (BKS.Cg-Dock7 m /+ Lepr db /+J) were obtained from Charles River laboratories and bred in our animal facility.

Techniques: Control, Imaging, MANN-WHITNEY