Figure 5.

Piezo1 channel knockdown by gene silencing with short hairpin RNA (shRNA) reduced the functional expression of Piezo1 not Piezo2 channels. (A–D) Immunoreactivity to the Piezo1 channels (green in A,C) or the Piezo2 channels (red in B,D) in human odontoblasts transfected by shRNA including a vector specific for human Piezo1 (C,D), or including an empty vector control (A,B). Nuclei are shown in blue. Scale bar: 100μm. No fluorescence was detected in the negative controls (not shown). (E) Bar graph showing the percentage area of the Piezo1 channel-immunopositive cells (%; green columns), and those of Piezo2 channel-immunopositive cells (%; red columns) in immunofluorescence analysis from human odontoblasts transfected by shRNA including a vector specific for human Piezo1 (second left and most right columns), or including an empty vector control (most left and third left). Each bar indicates the mean±SD of five experiments. (F) Representative traces of transient increases in [Ca²⁺]_i during mechanical stimulations induced by vertical micropipette displacement downward by 8.0μm (black boxes at the top) in standard extracellular solution in cells transfected by shRNA including a vector specific for human Piezo1 (shRNA-Piezo1 transfected), or including an empty vector control (shRNA-Control transfected). (G) Bar graph of the values of [Ca²⁺]_i increases induced by mechanical stimulation (8.0μm) in cells transfected by shRNA including a vector specific for human Piezo1 (gray column), or including an empty vector control (open column). The resting value is shown as F/F₀=1.0. The numbers in parentheses indicate the number of cells tested. Asterisks denote statistically significant differences between columns (shown by solid line in G): ^*p<0.05; N.S., not significant.