Journal: bioRxiv

Article Title: Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

doi: 10.1101/2023.02.23.529257

Figure Lengend Snippet: Kinase inhibitor screening reveals PI4KIIIβ as modulator of bafilomycin-induced EV secretion. a) Correlation plot depicting normalized HA-NL-CD63 secretion for individual kinase inhibitors from the kinase inhibitor screen performed under basal and bafilomycin-induced conditions. Grey area corresponds to the mean ± 2 times the standard deviation of the DMSO controls under bafilomycin-induced conditions. Kinase inhibitors in the green area were considered selective inhibitors of bafilomycin-induced HA-NL-CD63 secretion. b) Effect of PI4KIIIβ inhibition (PI4KIII Beta inhibitor 3, 1 μM) on bafilomycin- induced HA-NL-CD63 secretion. Data represents the mean ± S.E.M of four independent experiments. **P<0.01 using a one- sample, two-tailed T-test. c) Dose-inhibition curve of the PI4KIII Beta inhibitor 3 on bafilomycin-induced HA-NL-CD63 secretion. Data is a representative of three independent experiments. The IC 50 value of 6.4 ±1.5 nM (mean + S.E.M) was determined based on three independent experiments. d) Confirmation of PI4KIIIβ knockout at the protein level in HA-NL- CD63-CRISPR HEK293 cells. e) Effect of PI4KIIIβ KO on HA-NL-CD63 secretion under basal and bafilomycin-induced conditions. Data represents the mean ± S.E.M of three independent experiments. **p <0.01 using a two-sample, two-tailed Student’s T test. f) Western blot against EV marker proteins on cell lysates and isolated small EVs from control and bafilomycin-induced HEK293 cells in the presence or absence of PI4KIIIβ inhibitor (PI4KIII Beta inhibitor 3, 1 μM). Representative of two independent experiments. g) Effect of PI4KIIIβ inhibition (PI4KIII Beta inhibitor 3, 1 μM) on sEV secretion as determined by Nanoparticle Tracking Analysis. Representative of four independent experiments. h) Quantification of (g). Combined data of four independent experiments. Data is color-coded by biological replicate, where dots represent each biological replicate, and the horizontal bars represent mean ± SEM. **, P < 0.01 using a Student’s two-tailed two-sample t-test paired by biological replicate. i) Effect of PI4KIIα (PI-273, 2 μM) and PI4KIIIα (10 nM, GSK-A1) inhibition on HA-NL-CD63 secretion under basal and bafilomycin-induced conditions. Data represents the mean ± S.E.M of three (PI4KIIα) or four (PI4KIIα) independent experiments.

Article Snippet: U18666A (cat. no. U3633), Ionomycin (cat. no. IO634) and GSK-A1 (cat. no. SML2453) were obtained from Merck.

Techniques: Standard Deviation, Inhibition, Two Tailed Test, Knock-Out, CRISPR, Western Blot, Marker, Isolation

Journal: The Journal of Cell Biology

Article Title: Phosphatidylserine synthesis controls oncogenic B cell receptor signaling in B cell lymphoma

doi: 10.1083/jcb.202212074

Figure Lengend Snippet: PTDSS1 controls the PI4P pool at the PM to regulate BCR-derived signaling. (A) IP 1 production in Ramos cells. The cells were cultured for 3 d with 100 nM PTDSS1i and then stimulated with the indicated concentrations of anti-IgM F(ab’) 2 in the presence of LiCl. Data are presented as the mean + SD of three independent experiments. Statistical P values based on Welch’s t test are shown. (B) PIP levels in Ramos cells cultured for 3 d with vehicle or 100 nM PTDSS1i. Data are presented as the mean ± SD of three independent experiments with a dot plot of values for the individual experiments. Statistical P values based on one-way ANOVA followed by Šídák’s multiple comparisons test are shown. (C) Intracellular distribution of PI4P in vehicle- or PTDSS1i-treated Ramos cells. Ramos cells stably expressing EGFP-2xP4M SidM were cultured for 24 h with 100 nM PTDSS1i and then fluorescent images were analyzed using confocal microscopy (left). Fluorescence intensity ratio of PM to cytosol (PM/Cyto.) was quantified and dot-plotted for each cell (right). Statistical P values based on Mann–Whitney U test are shown. The scale bar represents 5 μm. (D) The effects of PI4KIIIα inhibitor (A1) on the intracellular distribution of PI4P and PI(4,5)P 2 in PTDSS1i-treated Ramos cells. Ramos cells stably expressing EGFP-2xP4M SidM or mTagBFP2-PH PLCδ1 were cultured for 24 h with 100 nM PTDSS1i followed by treatment with 100 nM A1 for 60 min and then fluorescent images were analyzed using confocal microscopy (left). Fluorescence intensity ratio of PM to cytosol (PM/Cyto.) was quantified and dot-plotted for each cell (right). Statistical P values based on one-way ANOVA followed by Kruskal–Wallis multiple comparisons test are shown. Scale bar represents 5 μm. (E and F) The effects of A1 treatment on enhanced BCR signaling in PTDSS1i-treated Ramos cells. PTDSS1i-treated cells pre-treated with 100 nM A1 for 60 min were stimulated with 20 μg/ml anti-IgM F(ab’) 2 and then IP 1 production (E) and Ca 2+ responses (F) were analyzed. Data are presented as the mean ± SD of three independent experiments. Statistical P values based on one-way ANOVA followed by Šídák’s multiple comparisons test are shown.

Article Snippet: Antibodies and reagents were purchased by vendors and used at the indicated dilution or concentration as follows: rabbit monoclonal anti-PTDSS1 (1:1,000, Cat# ab157222; Abcam), mouse monoclonal anti-α-tubulin (1:2,000, Cat#T6199; Sigma-Aldrich), rabbit polyclonal anti-Nir2 antibody (1:500, Cat#HPA003978; Sigma-Aldrich), rabbit polyclonal anti-ORP8 antibody (1:500, Cat#99069; Abcam), mouse monoclonal anti-KDEL antibody (1:1,000, Cat#ADI-SPA-827; Enzo Life Sciences), mouse monoclonal anti-GM130 antibody (1:500, Cat#610822; BD Biosciences), anti-GS28 antibody (1:500, Cat#611184; BD Biosciences), rabbit polyclonal anti-COXIV antibody (1:500, Cat#PM063; MBL), mouse monoclonal anti-SNX1 antibody (1:500, Cat#611482; BD Biosciences), mouse monoclonal anti-TfnR antibody (1:500, Cat#ab38171; Abcam), FITC-conjugated mouse monoclonal anti-human IgM (1:200, Cat# 314506; BioLegend), PE-conjugated mouse monoclonal anti-human CD79B (1:500, Cat# 341404; BioLegend), PE-conjugated mouse monoclonal anti-human CD19 (1:1,000, Cat# 555413; BD Biosciences), PE-conjugated mouse monoclonal anti-human CD79B (1:500, Cat# 341404; BioLegend), APC-conjugated mouse monoclonal anti-human Ig light chain λ (1:200, Cat# 316610; BioLegend), Alexa Fluor 647-conjugated mouse monoclonal anti-human CD22 (1:500, Cat# 302517; BioLegend), PE-conjugated mouse monoclonal anti-pY759-PLCγ2 (1:6, Cat# 558490; BD Biosciences), PE-conjugated mouse monoclonal anti-pY84-BLNK (1:6, Cat# 558442; BD Biosciences), PE-conjugated mouse monoclonal anti-pY352-Syk (1:20, Cat# 683704; BioLegend), goat anti-human IgM F(ab’) 2 (Cat# 397302; BioLegend), HRP-conjugated donkey anti-rabbit IgG F(ab’) 2 (1:10,000, Cat#NA9340; Cytiva), HRP-conjugated sheep anti-mouse IgG F(ab’) 2 (1:10,000, Cat#NA9310; Cytiva), NucView488 (1 μmM, Cat#10402; Biotium), U73122 (10 μmM, Cat#ab120998; Abcam), and GSK-A1 (100 nM, Cat#SML2453; Sigma-Aldrich).

Techniques: Derivative Assay, Cell Culture, Stable Transfection, Expressing, Confocal Microscopy, Fluorescence, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Phosphatidylserine synthesis controls oncogenic B cell receptor signaling in B cell lymphoma

doi: 10.1083/jcb.202212074

Figure Lengend Snippet: Lipid transporters mediate BCR regulation by PTDSS1. (A) Schematic of phospholipids transfer at the ER–PM contact site. (B) The effect of A1 on intracellular localization of mCherry-ORP8 in PTDSS1i-treated Ramos cells. Ramos cells stably expressing mCherry-ORP8 were cultured for 24 h with 100 nM PTDSS1i and then treated with 100 nM A1. Fluorescent images were analyzed at the indicated time points using confocal microscopy. Scale bar represents 5 μm. (C and D) PIP levels in ORP5/8-DKO or Nir2/3-DKO Ramos cells in the absence (left) or presence (right) of 100 nM PTDSS1i. Data are presented as a dot plot of values for the individual biological replicates. Statistical P values based on one-way ANOVA followed by Šídák’s multiple comparisons test are shown. (E) BCR-induced Ca 2+ responses of ORP5/8-DKO or Nir2/3-DKO Ramos cells in the absence (left) or presence (middle) of 100 nM PTDSS1i. Ramos cells pretreated with or without 100 nM PTDSS1i for 24 h were stimulated with 20 μg/ml anti-IgM F(ab’) 2 . The values for area under curve (A.U.C.) are shown (right panel). Data are presented as the mean ± SD of three independent experiments. Statistical P values based on one-way ANOVA followed by Šídák’s multiple comparisons test are shown. (F and G) PL levels in ORP5/8-DKO or Nir2/3-DKO Ramos cells in the absence or presence of 100 nM PTDSS1i. Relative abundance for PS, PI, and PA are presented as a percentage of the values of the vehicle-treated control group. Each dot indicates the value for the individual biological replicates. Statistical P values based on one-way ANOVA followed by Šídák’s multiple comparisons test are shown.

Article Snippet: Antibodies and reagents were purchased by vendors and used at the indicated dilution or concentration as follows: rabbit monoclonal anti-PTDSS1 (1:1,000, Cat# ab157222; Abcam), mouse monoclonal anti-α-tubulin (1:2,000, Cat#T6199; Sigma-Aldrich), rabbit polyclonal anti-Nir2 antibody (1:500, Cat#HPA003978; Sigma-Aldrich), rabbit polyclonal anti-ORP8 antibody (1:500, Cat#99069; Abcam), mouse monoclonal anti-KDEL antibody (1:1,000, Cat#ADI-SPA-827; Enzo Life Sciences), mouse monoclonal anti-GM130 antibody (1:500, Cat#610822; BD Biosciences), anti-GS28 antibody (1:500, Cat#611184; BD Biosciences), rabbit polyclonal anti-COXIV antibody (1:500, Cat#PM063; MBL), mouse monoclonal anti-SNX1 antibody (1:500, Cat#611482; BD Biosciences), mouse monoclonal anti-TfnR antibody (1:500, Cat#ab38171; Abcam), FITC-conjugated mouse monoclonal anti-human IgM (1:200, Cat# 314506; BioLegend), PE-conjugated mouse monoclonal anti-human CD79B (1:500, Cat# 341404; BioLegend), PE-conjugated mouse monoclonal anti-human CD19 (1:1,000, Cat# 555413; BD Biosciences), PE-conjugated mouse monoclonal anti-human CD79B (1:500, Cat# 341404; BioLegend), APC-conjugated mouse monoclonal anti-human Ig light chain λ (1:200, Cat# 316610; BioLegend), Alexa Fluor 647-conjugated mouse monoclonal anti-human CD22 (1:500, Cat# 302517; BioLegend), PE-conjugated mouse monoclonal anti-pY759-PLCγ2 (1:6, Cat# 558490; BD Biosciences), PE-conjugated mouse monoclonal anti-pY84-BLNK (1:6, Cat# 558442; BD Biosciences), PE-conjugated mouse monoclonal anti-pY352-Syk (1:20, Cat# 683704; BioLegend), goat anti-human IgM F(ab’) 2 (Cat# 397302; BioLegend), HRP-conjugated donkey anti-rabbit IgG F(ab’) 2 (1:10,000, Cat#NA9340; Cytiva), HRP-conjugated sheep anti-mouse IgG F(ab’) 2 (1:10,000, Cat#NA9310; Cytiva), NucView488 (1 μmM, Cat#10402; Biotium), U73122 (10 μmM, Cat#ab120998; Abcam), and GSK-A1 (100 nM, Cat#SML2453; Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, Cell Culture, Confocal Microscopy