Fig. 6
TMEM106B induces lysosomal exocytosis. a 393P cells with inducible expression of GFP-TMEM106B or GFP alone were transfected with an RFP-LAMP1 construct. After induction for 24–48 h, cells were counter stained with Hoechst33342. Live cells were imaged in real time. Indicated scale bar = 5 µm. b Western blot analysis upon subcellular fractionation of cells with induction of GFP-TMEM106B or GFP control. c Immunofluorescence staining for endogenous LAMP1 on outer plasma membrane of cells. 344SQ cells induced for either GFP only as control or GFP-tagged TMEM106B were first fixed using paraformaldehyde followed by immune staining using a phycoerythrin (PE)-conjugated LAMP1 primary antibody, without permeabilization, to specifically stain the endogenous LAMP1 present on the cell surface. d Western blot analysis for secreted lysosomal proteins in the conditioned media of cells expressing GFP-TMEM106B or GFP. e Cathepsin B activity measured using specific activity assay, in 50 µl of conditioned media from cells with either TMEM106B induction or knockdown, as indicated. f Cathepsin D activity measured using specific activity assay, in 50 µl of conditioned media from cells with induction or knockdown of TMEM106B expression, as indicated. g Invasion of non-invasive 393P cells when suspended in conditioned media from cells induced for GFP control or TMEM106B. All asterisks indicate statistical significance by t test (n ≥ 3, *p ≤ 0.05)
