Rudhira depletion deregulates developmental endothelial TGFβ signaling.

(A) KEGG pathway map indicating TGFβ pathway genes from KEGG database that are deregulated upon rudhira depletion in mouse embryonic yolk sac, based on [14]. 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) NS and KD SVEC cells, transfected with constitutively active TGFβRI for 24h, were analyzed for Smad2 phosphorylation by Immunoblotting. Graph shows the quantitation of pSmad2/Smad2 levels (N= 3 independent experiments). (C, D) Control (NS), rudhira knockdown (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) qRT-PCR analysis of mmp9 and pai1, known Smad2/3 targets in ECs (N= 3 independent experiments). (E, E’) Immunostaining for phosphorylated Smad2/3 in control and rudhira conditional knockout (rudhCKO) embryos at E10.5. Boxed region in the embryonic heart in (E) marking the endocardium and the myocardium are 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.

Rudhira functions downstream of TGFβ receptor activation to promote TGFβ-dependent cell migration.

(A) Control (NS), rudhira knockdown (KD) and rescued KD (KD + Rudhira-2A-GFP) cells were analyzed for migration rates with or without TGFβ using a transwell migration assay. Graph shows the extent of cell migration as measured by crystal violet absorbance (N= 3 independent experiments). (B, C) HEK293 cells transfected with vector alone or Rudhira were analyzed for migration rates using a transwell migration assay in various conditions, as indicated. Graph shows the extent of cell migration as measured by crystal violet absorbance (N= 3 independent experiments). Statistical analysis was performed using one-way ANOVA. Error bars indicate SEM. Scale bar: (A, B) 100 μm. *p<0.05, **p<0.01, ***p<0.001.

Rudhira is essential for the release of Smads from microtubules for TGFβ pathway activation.

(A) Analysis of interaction between Rudhira, Smad2/3 and MTs by co-immunoprecipitation and immunoblotting, as indicated (N= 3 independent experiments). (B) Loss of Smad-MT interaction on TGFβ stimulation in SVEC cells (N= 3 independent experiments). (C) NS and KD cells were analyzed for MT and Smad2/3 interaction by PLA for Tubulin and Smad2/3 with or without TGFβ treatment. PLA dots represent Smad2/3 and β-tubulin interaction. Graph shows the quantitation of PLA dots per cell (quantitation from >80 cells for two knockdown cell lines from N= 3 independent experiments; also see Fig. S3). (D, E) Association of Rudhira and MTs was analyzed by immunostaining (D) or Proximity Ligation Assay (PLA) (E) for Rudhira and Tubulin with or without TGFβ stimulation in SVEC. PLA dots represent Rudhira and β-tubulin interaction. Graph (D) shows the quantitation of colocalization percentage (quantitation from ∼40 cells from 9 images each with or without TGFβ respectively from N= 3 independent experiments) and graph (E) shows the quantitation of PLA dots per cell (quantitation from 86 or 68 cells from 9 images each with or without TGFβ respectively from N= 3 independent experiments). Statistical analysis was performed using one-way ANOVA. Error bars indicate SEM. Scale bar: (C) 20 μm; (D, E) 10 µm. *p<0.05, **p<0.01, ***p<0.001.

Rudhira is a Smad2/3-dependent target of TGFβ signaling.

(A) Smad-binding element (SBE) matrix (SMAD3, ID MA0795.1; Smad4, ID MA1153.1) and predicted binding sites in mouse (in red) and human (in blue) rudhira promoters, from JASPAR and PSCAN bioinformatics tool. (B-D) TGFβ stimulation followed by qRT-PCR (B), immunoblot (C) or immunostaining (D) analysis (quantitation from 34 cells in each condition) of Rudhira levels in SVEC cells. Graphs in (C) and (D) show the quantitation of Rudhira levels with or without TGFβ (N= 3 independent experiments). (E) Analysis of Rudhira levels upon smad2 or smad3 knockdown in HEK293T cells by immunoblotting (N= 3 independent experiments). Statistical analysis was performed using one-way ANOVA. Error bars indicate SEM. Scale bar: (D) 10 µm. *p<0.05, **p<0.01, ***p<0.001.

TGFβ-dependent rudhira transcription stabilizes MTs.

(A) Analysis of Rudhira levels and MT stability (marked by Glu-Tubulin) upon treatments as indicated, by immunoblotting. Graphs show the quantitation of Rudhira or Glu-Tub levels (N= 3 independent experiments). (B) Analysis of MT resistance to Nocodazole mediated depolymerization at indicated dosage and time (N= 30 cells). (C) MT stability in serum starved NS or KD SVEC cells kept untreated or treated with TGFβ was analyzed by immunoblot for Glu-Tubulin. Graph shows the quantitation of Glu-Tub levels (N= 3 independent experiments). (D) Immunostaining for Glu-Tubulin in control and rudhira conditional knockout (rudhCKO) embryos at E10.5. Endocardium in the heart is marked by PECAM1. Boxed region in the embryonic heart is magnified in the insets. Red arrowheads in the insets mark the PECAM1 positive cells (N= 4 embryos for each genotype). Graph shows the quantitation of Glu-Tub fluorescence intensity in the endocardium. (E) Model depicting cellular and molecular response to TGF⍰ addition. Rudhira has an early and a late effect on MTs. Previously known information about Rudhira is shown in blue. Events identified in this study are shown in red or pink. Parallel lines indicate protein-protein interaction. Solid lines indicate direct effects. Dotted lines indicate effects that may be direct or indirect. Arrows indicate positive regulation. Bar-headed line indicates inhibition. Statistical analysis was performed using one-way ANOVA. Error bars indicate SEM. Scale bar: (B) 10 µm; (D) 100 µm. *p<0.05, **p<0.01, ***p<0.001.

(related to Fig. 1). Cell line validation.

(A, B) Validation of Rudhira knockdown and restoration in SVEC cells by immunoblotting. 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.

(related to Fig. 1). Rudhira acts downstream to TGFβ receptors.

(A) qRT-PCR analysis of tgfβrI and tgfβrII in Rudhira knockdown endothelial cells (N= 3 independent experiments). (B) TGFβRI phosphorylation was analyzed using immunoblot technique in SVEC NS and KD. Graph shows the quantitation of pTGFβRI/TGFβRI levels (N= 3 independent experiments). (C) NS and KD SVEC cells, transfected with constitutively active TGFβRI for 24h, were analyzed for total TGFβRI level by qRT-PCR. (D) SVEC control (NS) and knockdown (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 (rudhCKO) embryos at E10.5. Boxed region in the embryonic heart in (E) marking the endocardium and the myocardium are 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.

(related to Fig. 1, 2 and 3). Rudhira specifically regulates TGFβ-dependent Smad2/3 activation.

(A) SVEC cells treated with SB431542 (10µM) for 2h and analyzed for Smad2 phosphorylation by Immunoblotting. (B) NS and two KD SVEC lines (KD1 and KD2) were analyzed for MT-SMAD interaction by PLA for β-Tubulin and Smad2/3 With or without TGFβ treatment (N= 3 independent experiments). (C) qRT-PCR analysis of negative regulators of TGFβ pathway (N=3 independent experiments). Statistical analysis was performed Using One way ANOVA. Error bars indicate standard error of mean (SEM). Scale bar (B): 20 μm. *p<0.05, **p<0.01, ***p<0.001.

(related to Figs. 4 and 5). TGFβ induces rudhira transcription for MT stability.

(A) Transcription factor binding sites (TFBS) available from JASPAR and PSCAN bioinformatics tool(B) Analysis of Rudhira levels and MT stability (marked by Glu-Tubulin) upon treatments as indicated by immunoblotting (N= 3 independent experiments). (C) Analysis of MT stability in serum starved WT and KD cells kept untreated or treated with TGFβ or serum (N= 3 independent experiments).

Primers used for qRT-PCR analysis.

Predicted Transcription factor binding sites in mouse and human rudhira/BCAS3 promoters, obtained from JASPAR bioinformatics tool.

The oligonucleotide sequence of the smad2 and smad3 shRNAs.