Fig. 1: RSPO2 and RSPO3 antagonize BMP4 signaling WNT independently.
From: R-spondins are BMP receptor antagonists in Xenopus early embryonic development
a, b BRE reporter assay in HEPG2 cells upon siControl (a) or siβ-catenin (b) transfection, with or without overnight BMP4 and RSPO1-4 treatment as indicated. n = 3 biologically independent samples. c, d Western blot analyses of phosphorylated Smad1 (pSmad1) and total Smad1 (tSmad1) in HEPG2 cells stimulated by BMP4, treated with or without increasing amount of RSPO2 (c) or RSPO3 (d) overnight. Cells were starved 3–6 h before the stimulation. Ratio, relative levels of pSmad1 normalized to tSmad1. Representative data from two independent experiments are shown. e qRT-PCR analysis of BMP target ID1 in HEPG2 cells upon BMP4, with or without overnightRSPO2 treatment. n = 3 experimentally independent samples. Data are displayed as means ± SD. **P < 0.01, ***P < 0.001 from two-tailed unpaired t-test. f, g BRE reporter assay in HEPG2 cells upon siLRP5/6 and siLGR4/5 knockdowns, with or without overnight BMP4 and RSPO2 treatment as indicated. n = 3 biologically independent samples. h BRE reporter assay in HEPG2 cells stimulated overnight by BMP4 with or without increasing amount of WNT3A treatment. WNT3A activity was validated in Supplementary Fig. 1b. n = 3 biologically independent samples. i Domain structures of RSPO2 and deletion mutants used in j. sp, signal peptide; FU, furin domain; TSP1, thrombospondin domain 1. j BRE reporter assay in HEPG2 cells stimulated overnight with BMP4, and with or without RSPO2 WT or FU1/2 or TSP1 deletion mutants, respectively. n = 3 biologically independent samples. Data for reporter assays (a, b, f–h, j) are displayed as means ± SD, and show a representative of multiple independent experiments. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 from two-tailed unpaired t-test (a, b, f, g, j) or one-way ANOVA with Dunnett test (h). k, l Western blot analyses of phosphorylated Smad1 (pSmad1) and total Smad1 (tSmad1) in H1581 cells upon siRNA transfection as indicated, with 0 h, 1 h, and 2 h of BMP4 stimulation. Ratio, relative levels of pSmad1 normalized to tSmad1. Representative data from two independent experiments are shown.
