Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: A-C Fibroblasts were fixed 10 h after wound scratch and subjected to immunofluorescence analysis. Pard3 was visualized with Alexa Fluor 647 (A). Junctional organization was quantified by lacunarity (B) and average puncta size (C)., ****P < 0. 0001, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 4. (D-E) Pard3 protein expression levels were analyzed by western blot, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 5. (F) Co-localization of Pard3 and the junctional protein ZO-1 was assessed by co-immunofluorescence staining. Pard3 was visualized with Alexa Fluor 647 (red), ZO-1 with Alexa Fluor 488 (green), and nuclei with DAPI(Blue). (G) Schematic of the pLV-Pard3-GFP lentiviral construct driven by the Ubc promoter. Construct integrity was confirmed by sequencing and western blot analysis. (H) Fibroblasts with shRNA-mediated knockdown of NDR1/2 were infected with Pard3- GFP–expressing lentivirus and enriched by FACS. Pard3 localization was visualized by GFP in both live-cell and fixed-cell imaging. The white dashed line indicates the scratch wound edge. Scale bars in (A, F, H): 20 µm.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Immunofluorescence, shRNA, Expressing, Western Blot, Staining, Construct, Sequencing, Knockdown, Infection, Imaging

Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: (A) Schematic of Pard3 (top) highlighting the consensus NDR kinase phosphorylation motif (H.R..[S/T]); The N- terminal region of Pard3 (tagged with Myc at the N-terminus and 6×His at the C- terminus) contains the consensus phosphorylation site at Ser144. Wild-type (WT) and S144A mutant constructs were cloned into the pET22b backbone for inducible expression in E. coli DE3 cells (bottom). (B) Fibroblasts stably expressing lentivirus encoding GFP alone were subjected to wound-healing assays as controls; ****P < 0.0001; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40. (C-F) Validation of Pard3 phosphorylation at Ser144 by in vitro kinase assays. NDR1 and NDR2 (WT or kinase-dead [KD], , K118A for NDR1 and K119A for NDR2) were purified from HEK293T cells and incubated with purified WT N-Pard3 or S144A-mutant N-Pard3 protein. Reactions were performed in the presence of ATP-γ-S, and thiophosphorylation was detected by western blot using an anti–thiophosphate ester antibody, ***P < 0.001, **P < 0.01; One-Way ANOVA followed by Dunnett’s post hoc tests, n = 3. (G-H) Rescue wound-healing assays were performed to evaluate the effect of exogenous Pard3 or the Pard3-S144A mutant on fibroblast migration following NDR1/2 knockdown over 20 h, ****P < 0.0001, ***P < 0.001, *P < 0.05; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40-46.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Phospho-proteomics, Mutagenesis, Construct, Clone Assay, Expressing, Stable Transfection, Biomarker Discovery, In Vitro, Purification, Incubation, Western Blot, Migration, Knockdown

Journal: Neurobiology of disease

Article Title: Pleiotropic effects of mutant huntingtin on retinopathy in two mouse models of Huntington’s disease

doi: 10.1016/j.nbd.2024.106780

Figure Lengend Snippet: Summary of distinct pathological characteristics observed in retina of two HD mouse models. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; IPL: inner plexiform layer; ARR3: cone arrestin-3; ARR1: rod arrestin; GNAT1: rod transducin alpha-subunit; OLM: outer limiting membrane; ZO-1: tight junction protein, zonula occludens-1; Crb2: crumbs cell polarity complex component 2; Par3: cell polarity protein, partitioning-defective 3.

Article Snippet: Primary antibodies against the following proteins were used: PHP1 (1:800, MABN2490, MilliporeSigma), cone arrestin Lumi-J (1:500, gift from Dr. Cheryl Craft, University of Southern California), ZO-1 (1:500, 61–7300, ThermoFisher Scientific), Crb2 (1:400, BS-14046R, ThermoFisher), Par3 (1:400, NBP1–88861, Novus Biologicals), Centrin 2 (1:200, 15,877–1-AP, Proteintech), GNAT1 (1:2000, PA5–28336, ThermoFisher), rabbit C10C10 antibody against ARR1(1:500) , mouse monoclonal anti-rhodopsin antibody 1D4 ( ) (sc-57,432, Santa Cruz Biotechnology).

Techniques: Membrane, Transgenic Assay, Knock-In, Labeling, Expressing, Translocation Assay, Disruption, Staining