Kinesore-induced projections contain both tubulin and F-actin. HAP1 cells were treated with kinesore (100 µM) for 1 h and fixed with either ice-cold methanol (A) or 4% PFA (B). (A) Methanol-fixed cells were stained with antibodies against tubulin (TUB 2.1, detected by anti-mouse Alexa Fluor 568) or actin (rabbit anti–β-actin (1E35) directly conjugated with Alexa Fluor 488). Inset shows control cells treated with buffer/vehicle only. Scale bars on left and middle panels are 10 µm, and 5 µm on right panels. (B) PFA-fixed cells were stained with TUB2.1 and phalloidin–Alexa Fluor 488 to detect filamentous actin (F-actin). Boxed regions show cellular projections at increasing magnification and highlight overlapping patterns in actin and tubulin staining. Scale bars on left and middle panels are 10 µm, and 5 µm on right panels. (C) Kinesore-treated cells were briefly cold-treated before methanol fixation and antibody staining for actin and tubulin. Actin staining defines the projections in cold-treated cells. Scale bar is 10 µm.