PM repair in B cells depends on lysosomal exocytosis and ASM release. (A) Immunofluorescence images of LIMP2 staining in B cells. Bar, 2.5 µm. B cells were incubated with or without SLO for 5 min at 37°C and then stained for LIMP2, BCRs, and DNA at 4°C with (total) or without permeabilization (surface). (B) The mean fluorescence intensities (MFI) of LIMP2 staining on the surface of B cells with or without SLO exposure was measured by flow cytometry. Shown is the mean (± SD) of three independent experiments. (C) The activity of ASM released from B cells into the medium. B cells were exposed to SLO for 15 s at 37°C, and ASM activity in the supernatants was detected using an Amplex red Sphingomyelinase Assay kit and expressed as relative fluorescence units (RFU). Shown is the mean (± SD) of three independent experiments. (D) ASM in whole B cell lysates or secreted after treatment with SLO for 5 min at 37°C was detected by Western blotting with anti-ASM antibodies. (E) Secretion of lysosomal β-Hex from B cells treated with SLO for 5 min at 37°C was determined in triplicate using a fluorgenic substrate and expressed as a percentage of the total activity present in whole-cell lysates. (F) Percentage of B cells repaired after exposure to SLO in the presence or absence of the ASM inhibitor DPA. B cells were preincubated with 30 µM DPA for 30 min at 37°C before and during 5-min exposure to SLO, followed by PI staining and flow cytometry. (G) Comparison of the repair efficiency of B cells from wild-type (WT) or ASM knockout (KO) mice in the presence or absence of bacterial sphingomyelinase (SM). B cells were treated with SM (50 µM) during SLO exposure, followed by PI staining and flow cytometry. (H) Comparison of the repair efficiency of wild-type B cells in the presence or absence of Ca²⁺ and SM. Shown are the mean (± SD) of three to five independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001.