Functional analysis of LAMP2 and SORT1 knockdown in lenalidomide-resistant MM cells. (A) Western blotting for SORT1, LAMP2, and β-actin in cells with stable knockdown of SORT1 and LAMP2 via shRNA. Proteins were loaded at 15 μg per lane. (B) EV secretion was measured with an ExoScreen assay. Each signal value was normalized to that of the control (CTRL). (C) Illustration of the method used for the adhesion experiment. Seventy-two hours after seeding, cells were washed 5 times with phosphate-buffered saline (-), and the numbers of attached cells were counted. (D) Picture of KMS27R cells with LAMP2 or SORT1 knockdown. Bars represent 100 μm. (E) Cell counts of the adherent cells in panel D. (F) The viability of nonadherent cells was estimated by using trypan blue staining. The values were normalized to the total cell number. (G) Cell counts of the adherent cells using FN-coated plates. (H) Cell viability assay of KMS27R cells with LAMP2 or SORT1 knockdown in the presence of lenalidomide. The values were normalized to those of the negative control (0 μM lenalidomide). Top, normal plate; bottom, FN-coated plate. (I) Comparative analysis of KMS27R cells with LAMP2 or SORT1 knockdown cultured in normal and ultra-low attachment conditions via the cell viability assay. The signal values for the ultra-low attachment plate were normalized to those for the normal plate. The error bars indicate standard deviation values. *P < .05. Len, lenalidomide; n.s., not significant.