Journal: Developmental cell

Article Title: The Oncogenic PRL Protein Causes Acid Addiction of Cells by Stimulating Lysosomal Exocytosis.

doi: 10.1016/j.devcel.2020.08.009

Figure Lengend Snippet: Figure 3. Acid Addiction by Lysosomal Exocytosis (A) Indicated MDCK cell lines were cultured with 2 mg/mL DOX for 24 h. Fixed cells with or without membrane permeabilization were stained with anti-LAMP2 antibody and observed with a confocal laser scanning microscope. Bar, 20 mm. (B and C) Indicated cell lines were fixed and stained with anti-LAMP2 antibody without membrane permeabilization and subjected to FACS analyses. (B) A representative result of MDCK cell lines cultured with 2 mg/mL DOX for 24 h. A result using isotype control antibody is also indicated. (C) Percentage of surface LAMP2-positive cells (mean ± SEM, n = 2–5). The p values were determined either by Student’s two-tailed t tests (unpaired; HEK293 PRL3 #1 cells) or by one-way ANOVA with Holm-Sidak post hoc tests (MDCK PRL3 #1 cells and CNNM2/4-dKO cells). *p < 0.05, ****p < 0.0001. (D) HEK293 cells transfected with the indicated siRNAs were cultured for 48 h in pH-fixed medium (pH 6.5 or 7.5). Cell lysates were subjected to SDS-PAGE and immunoblotting with the indicated antibodies (left), or fixed cells were stained with anti-LAMP2 antibody without membrane permeabilization and subjected to FACS analyses (right). Data are shown as the percentage of surface LAMP2-positive cells (mean ± SEM, n = 4–5). The p values were determined by one-way ANOVA with Holm-Sidak post hoc tests. **p < 0.01. (E) Indicated HEK293 cell lines were fixed and stained with anti-LAMP2 antibody without membrane permeabilization and subjected to FACS analyses. Data are shown as the percentage of surface LAMP2-positive cells (mean ± SEM, n = 3–4). The p values were determined by one-way ANOVA with Holm-Sidak post hoc tests. **p < 0.01. See also Figures S2 and S4.

Article Snippet: cDNAs, Antibodies, and Chemicals cDNAs for WT and the C104S mutant form of murine PRL3, and anti-CNNM4 and anti-CNNM2 rabbit polyclonal antibodies were generated in the previous studies (Yamazaki et al., 2013; Funato et al., 2014; Funato et al., 2017).We also used the following commercially available antibodies: mouse monoclonal antibodies for LAMP2 (for MDCK and HEK293 cells, 1:100, Thermo, MA5-16561), LAMP1 (PE-conjugated, for B16 cells, 1:1000, BD Biosciences, 558661), PRL3 (1:500, Santa Cruz Biotechnology, sc-130355), a-tubulin (1:10000, Sigma, T9026), and b-actin (1:10000, Proteintech, 60008-1-Ig); rabbit polyclonal antibodies for NMHC-IIA (1:1000, Sigma, M8064), myosin light chain (MLC) 2 (1:500, Cell Signaling Technology, 3672), and phospho-MLC2 (Ser19, 1:500, Cell Signaling Technology, 3671), and chicken polyclonal antibody for GFP (1:5000, Abcam, ab13970).

Techniques: Cell Culture, Membrane, Staining, Laser-Scanning Microscopy, Control, Two Tailed Test, Transfection, SDS Page, Western Blot