Journal: bioRxiv

Article Title: Non-neuronal, TGF-β–driven extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model

doi: 10.1101/2025.08.17.670724

Figure Lengend Snippet: a, b, Immunoblot quantification of phosphorylated AKT (p-AKT^S473) and ERK1/2 (p-ERK1/2^T202/Y204) in MOs from HC and PSP-RS donors shows increased activation of both pathways in PSP-RS (mean ± s.e.m.; n = 3; P = 0.0269 for p-AKT, P = 0.0203 for p-ERK1/2; unpaired t-test with Welch’s correction). c, Expression of the PP2A phosphatase is significantly reduced in PSP-RS MOs (P = 0.0179). d, Treatment of PSP-RS MOs with the PI3K inhibitor LY294002 reduces p-AKT levels and diminishes phosphorylation of tau at S396 and T181 (P = 0.0081, P = 0.0051, and P = 0.0014, respectively). e, Inhibition of ERK1/2 signaling with PD0325901 leads to decreased p-ERK1/2 and reduced phospho-tau at S396 and T181 in PSP-RS MOs (P = 0.0204, P = 0.0103, and P = 0.0146, respectively). f, Pharmacological blockade of TGF-β signaling with SB431542 reduces p-AKT and p-ERK1/2, as well as phospho-tau at S396 and T181 in PSP-RS cultures (P = 0.0255, P = 0.0314, P = 0.0059, and P = 0.0167, respectively). Band intensities are normalized to total protein or histone H3 (H3) as loading controls. Uncropped western blot gels and statistics are available in Additional File 1-2.

Article Snippet: In PSP-RS organoids, TGFβ pathway inhibition was achieved using the selective ALK5 inhibitor SB431542 (10 μM; Miltenyi Biotec).

Techniques: Western Blot, Activation Assay, Expressing, Phospho-proteomics, Inhibition

Journal: bioRxiv

Article Title: Non-neuronal, TGF-β–driven extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model

doi: 10.1101/2025.08.17.670724

Figure Lengend Snippet: a, Representative images of F-actin staining (phalloidin, red) in MOs primary cultures from healthy controls (HC) and PSP-RS patients. DNA is counterstained in blue (DAPI). Insets show higher magnification of boxed areas. Scale bar, 50 µm. b, Quantification of mean fluorescence intensity (MFI) for F-actin reveals significantly increased actin polymerization in PSP-RS (mean ± s.e.m.; n = 8 images; *** P = 0.0002, unpaired t -test with Welch’s correction). c, Western blot showing reduced expression of actin-capping protein CapZ in PSP-RS compared to HC. d, Western blot of acetylated α-tubulin (Lys40) shows decreased levels in PSP-RS MOs. Uncropped western blot gels are available in Source Data e, Representative images and quantification of RhoA-GTP levels (green) show increased active RhoA in PSP-RS cells (mean ± s.e.m.; n = 60 cells HC, 61 cells PSP; **** P < 0.0001, Mann–Whitney test). DNA, DAPI (blue). Scale bar, 25 µm. f, Treatment of PSP-RS MOs with ROCK inhibitor Y-27632 reduces F-actin intensity (mean ± s.e.m.; n = 8 images HC, 7 images PSP; P = 0.0490, unpaired t -test with Welch’s correction). Scale bar, 50 µm. g, Exogenous TGF-β treatment of HC MOs increases F-actin intensity (mean ± s.e.m.; n = 23 ROIs HC, 24 ROIs HC+TGFβ; ** P = 0.0052, unpaired t -test with Welch’s correction). Scale bar, 50 µm. h, TGF-β I receptor inhibitor SB431542 reduces F-actin intensity in PSP-RS cultures (mean ± s.e.m.; n = 23 ROIs PSP, 26 ROIs PSP+SB; **** P < 0.0001, unpaired t -test with Welch’s correction). Scale bar, 50 µm. Statistics are available in Additional File 2.

Article Snippet: In PSP-RS organoids, TGFβ pathway inhibition was achieved using the selective ALK5 inhibitor SB431542 (10 μM; Miltenyi Biotec).

Techniques: Staining, Fluorescence, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Non-neuronal, TGF-β–driven extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model

doi: 10.1101/2025.08.17.670724

Figure Lengend Snippet: a, Representative confocal images of MOs from HC and PSP-RS donors stained for synaptophysin (SYP, red), PSD95 (green), and DNA (DAPI, blue) show reduced pre- and post-synaptic puncta in PSP-RS. Scale bar, 10 μm. b, Quantification of synaptic markers reveals decreased fluorescence intensity for SYP (P = 0.0273) and PSD95 (**** P < 0.0001), along with fewer synaptic elements per nucleus (P = 0.0362) in PSP-RS (mean ± s.e.m.). c, Expression of the dendritic spine marker drebrin is significantly reduced in PSP-RS MOs (mean ± s.e.m.; n = 17 images; P = 0.0210). Scale bar, 50 μm. d, Representative images of DA neurons stained for MAP2 (red) reveal diminished neurite complexity in PSP-RS cultures. Scale bar, 25 μm. e, Quantification shows significantly shorter dendritic branch length (*** P = 0.0007) and fewer branches per neuron (** P = 0.0074) in PSP-RS DA neurons (mean ± s.e.m.; n = 14–15 neurons). f, g, Treatment of PSP-RS neurons with the TGF-β receptor inhibitor SB431542 rescues dendritic complexity, increasing both branch length (**** P < 0.0001) and number of branches per neuron (*** P = 0.0005; n = 35 neurons). Scale bar, 25 μm. h, Exogenous TGF-β treatment of HC MOs reduces SYP and PSD95 puncta, mimicking the PSP-RS synaptic phenotype. Scale bar, 25 μm. i, Quantification confirms significant reductions in SYP (**** P < 0.0001), PSD95 (**** P < 0.0001), and synaptic elements per nucleus (*** P = 0.0005) following TGF-β treatment (mean ± s.e.m.; n = 30–32 regions of interest). Statistics are available in Additional File 2.

Article Snippet: In PSP-RS organoids, TGFβ pathway inhibition was achieved using the selective ALK5 inhibitor SB431542 (10 μM; Miltenyi Biotec).

Techniques: Staining, Fluorescence, Expressing, Marker

Journal: eLife

Article Title: Rudhira-mediated microtubule stability controls TGFβ signaling during mouse vascular development

doi: 10.7554/eLife.98257

Figure Lengend Snippet: ( A ) KEGG pathway map indicating TGFβ pathway genes from KEGG database that are deregulated upon rudhira depletion in mouse embryonic yolk sac, based on . Genes were mapped based on fold change in rudhira -/- yolk sacs in comparison to control to understand pathway regulation. Green: downregulated, red: upregulated. Blue circles indicate affected genes or processes known to be affected upon Rudhira depletion from earlier studies. ( B ) Non-silencing (NS) cells and knockdown (KD) saphenous vein endothelial cells (SVECs), transfected with constitutively active TGFBR1 for 24 hr, were analyzed for Smad2 phosphorylation by immunoblotting. Graph shows the quantitation of pSmad2/Smad2 levels (N=3 independent experiments). ( C, D ) Control (NS), rudhira KD, and rescued KD (KD+Rudhira-2A-GFP) cells were kept untreated or treated with TGFβ and used for various assays, as indicated. ( C ) Immunoblot for Smad2/3 and pSmad2. Graph shows the quantitation of pSmad2/Smad2 levels (N=3 independent experiments). ( D ) Quantitative RT-PCR (qRT-PCR) analysis of Mmp9 and Serpine , known Smad2/3 targets in endothelial cells (ECs) (N=3 independent experiments). ( E, E’ ) Immunostaining for phosphorylated Smad2/3 in control and rudhira conditional knockout ( Rudhira CKO ) embryos at E10.5. Boxed region in the embryonic heart in ( E ) marking the endocardium and the myocardium is magnified in ( E’ ) (N=3 embryos for each genotype). White dotted line indicates the endocardium. Graph shows the quantitation of pSmad2/3 levels (N=3 independent experiments). Statistical analysis was performed using two-way ANOVA ( A, B, C, D ) and Student’s t-test ( E ). Error bars indicate standard error of mean (SEM). *p<0.05, **p<0.01, ***p<0.001. Scale bar: ( E ) 100 µm; ( E’ ) 20 μm. Figure 1—source data 1. Prism files showing graphs and statistical analysis. Figure 1—source data 2. Raw uncropped, unedited blots. Figure 1—source data 3. Uncropped blots with relevant bands labelled.

Article Snippet: Constitutively active TGFBR1 (pcDNA3-ALK5 T204D, 80877) was purchased from Addgene for transfection of SVEC NS and KD cells.

Techniques: Comparison, Control, Knockdown, Transfection, Phospho-proteomics, Western Blot, Quantitation Assay, Quantitative RT-PCR, Immunostaining, Knock-Out

Journal: eLife

Article Title: Rudhira-mediated microtubule stability controls TGFβ signaling during mouse vascular development

doi: 10.7554/eLife.98257

Figure Lengend Snippet: ( A ) Quantitative RT-PCR (qRT-PCR) analysis of Tgfbr1 and Tgfbr2 in Rudhira knockdown (KD) endothelial cells (N=3 independent experiments). ( B ) TGFBR1 phosphorylation was analyzed using immunoblot technique in saphenous vein endothelial cell (SVEC) non-silencing (NS) and KD. Graph shows the quantitation of pTGFBR1/TGFBR1 levels (N=3 independent experiments). ( C ) NS cells and KD SVECs, transfected with constitutively active TGFBR1 for 24 hr, were analyzed for total TGFBR1 level by qRT-PCR. ( D ) SVEC control (NS) and KD cells were stimulated by BMP4 and used for immunoblotting of pSmad1/5/8. Graph shows the quantitation of pSmad1/5/8 by Smad1/5/8 levels (N=3 independent experiments). ( E, E’ ) Immunostaining for total Smad2/3 in control and rudhira conditional knockout ( Rudhira CKO ) embryos at E10.5. Boxed region in the embryonic heart in ( E ) marking the endocardium and the myocardium is magnified in ( E’ ) (N=3 embryos for each genotype). White dotted line indicates the endocardium. Graph shows the quantitation of Smad2/3 level (N=3 independent experiments). Statistical analysis was performed using one-sample t-test. Error bars indicate standard error of mean (SEM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar: ( E ) 100 µm; ( E’ ) 20 μm. Figure 1—figure supplement 2—source data 1. Prism files showing graphs and statistical analysis. Figure 1—figure supplement 2—source data 2. Raw uncropped, unedited blots. Figure 1—figure supplement 2—source data 3. Uncropped blots with relevant bands labelled.

Article Snippet: Constitutively active TGFBR1 (pcDNA3-ALK5 T204D, 80877) was purchased from Addgene for transfection of SVEC NS and KD cells.

Techniques: Quantitative RT-PCR, Knockdown, Phospho-proteomics, Western Blot, Quantitation Assay, Transfection, Control, Immunostaining, Knock-Out