PIEZO1 characterization in RBCs and in CD34⁺ blood cells during erythroid differentiation. (A) Laser-scanning confocal immunofluorescence images of a peripheral blood smear from a control subject stained with rabbit polyclonal antibody to PIEZO1 (red) and mouse monoclonal antibody to glycophorin A (membrane marker, green), showing colocalization of the 2 signals (merge), and a bright-field image of a red cell. Cells were imaged with a Leica TCS SMD FLIM confocal microscope equipped with a 1.4 NA oil-immersion HCX PL APO CS 100× objective. Luminosity and contrast were uniformly adjusted with Axiovision software. Representative of 3 independent experiments. (B) PIEZO1 mRNA levels (normalized to GADPH) in CD34⁺ cells induced to erythroid differentiation by EPO at 0, 7, and 14 days by quantitative reverse transcription PCR. *P value, .003 (CD34⁺ 14D vs CD34⁺ 7D). (C) Immunoblot of PIEZO1 protein in lysate of CD34⁺ cells induced to erythroid differentiation at 0, 7, and 14 days (50 μg of protein; GAPDH was loading control). The graph below shows densitomteric analysis of this and similar blots. (D) Laser-scanning confocal microscopy images of CD34⁺ cells induced to differentiation at 7 and 14 days by EPO, showing colocalization of PIEZO1 (red) and glycophorin A (green). Cells were imaged with a Leica TCS SMD FLIM confocal microscope equipped with a 1.4 NA oil-immersion HCX PL APO CS 63× objective. Bright-field images show the morphology of CD34⁺ cells after 7 or 14 days of erythroid differentiation. Luminosity and contrast were adjusted using Axiovision software. (E) PIEZO1 mRNA levels (normalized to GAPDH) in HEK-293 cells transiently transfected (72 hours) with empty vector or with cDNA encoding WT PIEZO1 or PIEZO1 mutants R2488Q and R2456H. Mean + SEM of 3 experiments. (F) Immunoblot of PIEZO1 polypeptide in HEK-293 cells transiently transfected (72 hours) with empty vector or with cDNA encoding WT PIEZO1 or PIEZO1 mutants R2488Q and R2456H. GAPDH serves as loading control. One of 2 similar experiments.