Extended Data Fig. 6: The TGFβ–SMAD pathway in the regulation of CCND3.
From: Reduced cyclin D3 expression in erythroid cells protects against malaria
a, Stimulation of SMAD3 (left panel) and CCND3 mRNA expression (right panel) by TGFβ in HUDEP-2 cells (black line) at 60’, 120’, 240’, and 300’ time points, quantified by RT–qPCR analysis using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. SMAD3 and CCND3 stimulation is abrogated by addition of the TGFβ specific inhibitor, SB-505124 (red line SB-505124 + TGFβ, blue line SB-505124 alone). The the mean ± s.e.m is shown (n = 3 biologically independent experiments at 60’ and 300’; n = 4 biologically independent experiments at 120’ and 240’ time points). b, Top, RT–qPCR analysis of CCND3 mRNA level in HUDEP-2 cells transduced with single guide gRNAs (sgRNAs) targeting the coding region of SMAD3 and SMAD2 (ΔSMAD2/3) or SMAD3 alone (ΔSMAD3) compared to cells transduced with Cas9 only, without sgRNA (CTRL). The CCND3 mRNA expression was normalized to GAPDH mRNA. The mean ± s.e.m is shown (n = 3; 3 biologically independent replicates). Statistical significance, calculated by two-sided t-test, is indicated by asterisks (*P < .05, ***P < .001). The statistical results for all comparisons are provided in Supplementary Table 3. Bottom, western blot assay showing Cyclin D3 downregulation in HUDEP-2 cells after gene-editing of the SMAD3/SMAD2 (ΔSMAD2/3) or SMAD3 (ΔSMAD3) coding region; human β-actin was run on the same gel as loading control. Bands in immunoblots were quantified using ImageJ software (https://imagej.nih.gov/ij/download.html) and the ratio between Cyclin D3 and β-actin in each sample was normalized relative to control sample of lane 1 (CTRL). For gel source data, see Supplementary Fig. 1. c, Schematic diagram of the TGFβ signalling pathway involved in the regulation of CCND3 expression. TGFβ ligand binds to its receptor TGFβRII, leading to phosphorylation of TGFβRI, which in turn phosphorylates SMAD2/3, enabling their translocation to the nucleus. Phosphorylated SMAD2/3 complexes (pSMAD2/3) bind to the CCND3 erythroid-specific enhancer around rs112233623, thereby activating CCND3 gene expression (considering NM_001760.5). Red star in the locus 6p21.1 indicates rs112233623. The statistical results for all comparisons are provided in Supplementary Table 3.
