Fig 2: Expression of glomerular proteins. Representative laser confocal images of kidney sections from 9-day old Foxc2 fl/fl;Pod-Cre+ and Foxc2 fl/fl mice using antibodies labeling (A) proteins involved in anchoring of podocytes (ITGB1/ß1-integrin, ILK/integrin linked kinase and CYR61/CCN1), (B) basement membrane proteins (COL4A3 and COL4A4), and (C) components of the foot processes (i.e., slit diaphragm proteins (NPHS1/nephrin and NPHS2/podocin), anchoring of slit diaphragm to cytoskeleton (TJP1/ZO-1, ACTN4/a-actinin 4, CD2AP/CD2-associated protein, and NES/nestin), and foot process coating (PODXL/podocalyxin)).

Fig 3: Silencing of CD34 expression in HMEC-1.(A) Representative histograms showing the intensity of anti-CD34 (left panel) or anti-podocalyxin (right panel) labeling of parental HMEC-1 compared to an isotype control (grey peak). (B) Quantitative PCR (qPCR) analysis of CD34 and PODXL mRNA levels and (C) flow cytometric analysis of CD34 protein expression on the membrane of HMEC-1 72 h after transfection with CD34-targeting (siCD34) or non-targeting (NT) (siNT) siRNA. Stimulation with VEGF induced CD34 plasma membrane expression in both siNT-treated and siCD34 treated HMEC-1. However, CD34 expression in siCD34-treated HMEC-1 was less than 30% as compared to expression levels in siNT-treated cells. *Significantly different from non-stimulated cells with P < 0.05.

Fig 4: Podocyte-specific deletion of IRE1a exacerbates podocyte injury in adriamycin (ADR) nephrosis.A Albuminuria was exacerbated in ADR-treated IRE1a KO mice, compared with ADR-treated control (Ctrl) littermates. There are 4 mice per group, except 5 mice in the KO ADR group. B Representative images of glomerular histology (PAS stain). Bar = 60 µm. C Glomerular matrix expansion, evaluated with a pixel-counting algorithm, was greater in IRE1a KO mice than in control animals after ADR injection. 25 glomeruli in 4–5 mice per group were analyzed. D Kidney sections were stained for podocalyxin and nephrin. Bar = 20 µm. E Compared with the ADR-treated control group, podocalyxin and nephrin fluorescence intensity was lower in treated IRE1a KO mice. 16–27 glomeruli in 4–5 mice per group were analyzed. F, G Podocalyxin (PDX) runs as a doublet or a smear due to various post-translational modifications; the entire podocalyxin signal was quantified. PDX expression was reduced in glomerular lysates of IRE1a KO mice (immunoblot). *P = 0.05, **P = 0.01, ***P = 0.001.

Fig 5: Lung distal progenitor cells differentiate in 3D explant cultures.(A) Whole-mount immunostaining for the alveolar cell markers SFTPC and RAGE at consecutive culture days. By day 2, SFTPC and RAGE are expressed by most cells. Day 6 epithelial branches comprise both bipotent progenitor (BP) cells (SFTPC+/RAGE+) and differentiated alveolar cells (AT1, SFTPC-/RAGE+ and AT2, SFTPC+/RAGE-) in an organotypic pattern. (B) Collagen I promotes AT1 cell differentiation, compared with Matrigel alone. Cell identities were scored on day 6. (C) Markers of differentiated AT1 and AT2 cells are transcriptionally upregulated by day 6 of culture, at levels comparable with E18.5 distal lung tissue. Expression values are normalized to culture day 0. Each box depicts one biological replicate, showing the mean value among two technical replicates; 27 tip cultures were used for each time point. (D) AT1 and AT2 cells in organoids are morphologically distinct and display apico-basal polarity. AT1 cells are elongated and often populate stalk regions. Upper row: AT1 cells display basolateral localization of the AT1 cell marker RAGE and apical localization of PODXL. Lower row: epithelial branches in organoids display adhesions typical of intact epithelia, such as tight junctions (ZO-1). The AT2 cell marker LAMP3 is localized to the apical secretory compartment of surfactant-producing cells. Scale bars: 20 µm. (B) Mean values are displayed; error bars represent S.D. Figure 2—source data 1.Counts of alveolar cell types in organoid culture, Matrigel vs. Collagen. Normalized expression values of alveolar markers.