Journal: EMBO Reports
Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection
doi: 10.1038/s44319-024-00072-2
Figure Lengend Snippet: ( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, p-p38, p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.
Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.
Techniques: Western Blot, Control, Quantitative RT-PCR, Transfection, Expressing, Luciferase, Incubation
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![BMP signaling in NPCs increases the ratio of young adult type B cells to type E cells (A) P0 NPC cultures maintained for 5 DIV in the presence of 10% fetal bovine serum (FBS) and then for 1 day without FBS and in the absence or presence of BMP2 (20 ng/ml) or <t>BMP6</t> (20 ng/ml) were subjected to RNA-seq analysis. (B) Volcano plots showing changes in gene expression induced by BMP2 or BMP6 treatment. Differentially expressed genes (DEGs) that were upregulated by BMP2 or BMP6 treatment (edgeR: p < 0.05, log2[fold change] > 0.585) are shown in pink, whereas those downregulated by BMP2 or BMP6 (upregulated in the control) (edgeR: p < 0.05, log2[fold change] < –0.585) are shown in light blue. (C) Gene-set enrichment analysis (GSEA) for E16.5 B-like versus E-like cells with DEGs found to be upregulated or downregulated by BMP2 or BMP6 in (B). BMP2- or BMP6-upregulated DEGs were enriched in B-like cells, whereas BMP2- or BMP6- downregulated DEGs were enriched in E-like cells. NES, normalized enrichment score; FDR, false discovery rate. (D) GSEA for BMP2- or BMP6-treated NPC cultures versus control cultures with adult type B cell (qNSC) and adult type E cell gene sets (Shah et al., 2018). Adult type B cell genes were enriched in BMP2- or BMP6-treated cultures compared with control cultures, whereas adult type E cell genes were enriched in control cultures compared with BMP2- or BMP6-treated cultures. (E) Scheme for virus infection experiments. In utero infection (IUI) was performed at E14.5 with the indicated lentivirus cocktail (FU-T2A-mScarlet-W and FU-GeneX-T2A- EGFP-W lentiviruses), and mice were subjected to immunohistofluorescence staining at P25–30. OE, overexpression. (F) The lentiviruses FU-T2A-mScarlet-W and FU-ALK2QD-T2A-EGFP-W were injected into the lateral ventricle of embryos at E14.5, and the resulting mice were subjected to immunofluorescence analysis at P28–30. Brain sections were stained for EGFP, mScarlet, GFAP, and S100β. Light blue and pink arrowheads indicate young adult type B cells and type E cells, respectively. Young adult type B cells were defined as GFAP + cells with apical attachment in the V-SVZ of the lateral wall, and type E cells as S100β + cells. Scale bars, 50 μm. (G) Proportions of young adult type B cells and type E cells among infected cells (control or ALK2QD overexpressing) in the V-SVZ determined from images as in (F). Cells positive for both mScarlet and EGFP were excluded from the analysis. Data are means ± SD ( n = 3 mice). *** p < 0.001, two-tailed Student’s t test. See also .](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_51/10__1101_slash_2024__05__12__593751/10__1101_slash_2024__05__12__593751___F5.large.jpg)
