Transgenic approach to investigate the role of CD236R in P vivax rosetting. (A) Experimental design of schizont P vivax rosetting assay with cultured RBCs (cRBCs) generated from CD34⁺ hematopoietic stem cells. The stable knockdown of CD236R (glycophorin C) is obtained using Sigma lentivector with GFP and shRNA against CD236R cassette expression. The 2 subsets of cells: CD236R knockdown and CD236R⁺ cells were separated by flow cytometry using GFP expression, 1 day before performing the rosetting assay with P vivax schizonts isolated by magnetic sorting. (B) Flow cytometry histograms showing CD236R expression in GFP-positive cells (CD236R knockdown cells [green line]), GFP-negative (CD236R⁺ cells [black line]), and unstained cells (gray line). (C) Plots showing schizont P vivax rosetting with cRBC with CD236R knockdown (CD236R KD) and without knockdown (CD236⁺) with bright field (BF), Hoechst, and GFP detection. (D) Frequency of schizonts with or without rosetting in the presence of CD236R knockdown cRBCs or of CD236⁺ cRBCs showing a significant difference in proportion of schizonts able to form rosettes between the 2 different types of cRBCs (CD236R KD and CD236⁺).