Fig. 7

Secreted NPM binds to TLR4 and the binding increases upon UV treatment. a Sketch figure of experimental plan. ShScr and ShNPM secreting hCmPCs were treated or not with 50mJ/cm² UV for 4h. The supernatants of secreting cells were then collected and used to culture hCmPCs knocked-down for NPM (shNPM), here referred as recipient cells. b Representative WB demonstrating a >70% knockdown of NPM1 expression in hCmPC infected with a lentivirus encoding a NPM1-specific shRNA sequence (ShNPM) compared to control (ShScr). Uncropped images of blots are shown in Additional file 1: Figure S1d. c Densitometric analysis of NPM expression levels normalised by β-actin (n = 3; ^***P < 0.001). d PLA images of knocked-down NPM cells, cultured in the supernatants of ShScr (left panels) and ShNPM (right panels) treated or not with UV (lower and upper panels, respectively). Lower panels represent zoomed in images of the original PLA images. PLA assay barely detected NPM/TLR4 interaction in recipient cells cultured with the medium derived from untreated ShNPM secreting cells. When recipient cells were kept in the medium of UV irradiated secreting ShScr cells the dots increased (lower left panel). As a negative control, the IgG specific for each antibody (i.e. normal rabbit IgG used as the control of TLR4 antibody) and normal mouse IgG for NPM antibody (right panels) revealed the specificity of NPM and TLR4 interaction, since no red dots were present in the PLA. Scale bar 10μm. e Bar graph of the quantification of the PLA of dots per nuclei (n = 4; ^*P < 0.05)