DEV037267 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Figure S1 -

  Fig. S1. Mouse glomerular endothelial cell (MGEC) characterization. (A) β-Gal⁺ staining; (B) Flk-1⁺ immunostaining; (C) Rhodamine-labeled Bandereia simplicifolia Lectin isoform 4 (BSL4)⁺ immunostaining; (D) CD31⁺ immunostaining; (E) WT1^- immunostaining and phase image; (F) α-smooth muscle actin (SMA)^- immunostaining and phase image. Scale bar: 50 µm.

• Supplemental Figure S2 -

  Fig. S2. Kidney macrophage number is not altered by Sema3a gene deletion. F4/80 immunostaining of macrophages in Sema3a^+/+ (3A+/+) and Sema3a^-/- (3A-/-) newborn kidneys demonstrating a similar density of interstitial macrophages (bottom panel, white arrowheads) and an absence of glomerular macrophages (top panel). Scale bars: 20 µm for the top panel; 50 µm for the bottom panel.

• Supplemental Figure S3 -

  Fig. S3. The localization of PAX2 and synaptopodin is not regulated by Sema3a gene dosage. (A,B) Immunostaining for PAX2 (green) and synaptopodin (red) in Sema3a^-/- (A) and Sema3a-overexpressing (B) newborn kidneys and their respective controls demonstrating similar PAX2 expression in the nephrogenic cortex and synaptopodin expression in the most mature glomeruli. Negative controls for immunostaining are shown in panel A. Scale bar: 20 µm.

• Supplemental Figure S4 -

  Fig. S4. Compensatory changes in Sema3b and Sema3e expression induced by modulation of the Sema3a gene dose. (A) qPCR of Sema3a to 3g in Sema3a^-/- kidneys, normalized to GAPDH and expressed as fold change from Sema3a^+/+ (3A+/+), showing that kidney Sema3e mRNA expression is 2-fold higher in Sema3a^-/- kidneys. Sema3a expression is minimal in Sema3a^-/- mice, as expected. (B) qPCR of Sema3a to 3g in newborn Sema3a-overexpressing kidneys (+dox), normalized to GAPDH and expressed as fold change from control kidneys (-dox) showing 2.8-fold higher Sema3b mRNA levels upon podocyte Sema3a overexpression. As expected, total kidney Sema3a mRNA does not change, as overexpression is podocyte specific. In A and B, the dotted line indicates no change in expression; values <1 indicate downregulation; values >1 indicate upregulation. * indicates P<0.05, Sema3a^+/+ and Sema3a^-/-; δ indicates P<0.05, newborn Sema3a-overexpressing kidneys (+dox) vs controls (−dox).