Journal: Journal of Extracellular Biology

Article Title: Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

doi: 10.1002/jex2.70112

Figure Lengend Snippet: GDP‐bound Rab18 accumulates in perinuclear endosomes and promotes SHH‐XLEV secretion. (A–C) Airyscan microscopy of NIH3T3 cells stably expressing SHHN‐EGFP following treatment with ZSTK474 (1 µM, 1 h; A, upper), LY294002 (50 µM, 8 h; A, lower), or SF1670 (250 nM, overnight; B), with quantification of peripheral distribution scores (PDS; C). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 20. (D–F) Airyscan microscopy of NIH3T3 cells expressing EGFP‐Rab18Q67L (GTP‐bound mimetic; D) or EGFP‐Rab18S22N (GDP‐bound mimetic; E), with PDS quantification (F). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 7–16. (G–I) Airyscan immunofluorescence microscopy of EGFP‐Rab18S22N–expressing cells stained for AP‐1 (G) or AP‐3 (H) at low (upper) and high (lower) magnifications, with colocalization analysis (I). Arrowheads indicate perinuclear Rab18S22N puncta. Scale bars, 20 µm. *** p < 0.001; Welch's t test; n = 10.

Article Snippet: EGFP‐Rab18 (Addgene #49550; RRID: Addgene_49550), EGFP‐Rab18Q67L (Addgene #49596; RRID: Addgene_49596), and EGFP‐Rab18S22N (Addgene #49597; RRID: Addgene_49597) were gifts from Marci Scidmore (Huang et al. ).

Techniques: Microscopy, Stable Transfection, Expressing, Immunofluorescence, Staining

Journal: Journal of Extracellular Biology

Article Title: Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

doi: 10.1002/jex2.70112

Figure Lengend Snippet: Hsp90α and nSMase2 are effectors of GDP‐bound Rab18. (A) GST pull‐down assays using mouse brain lysates incubated with GST‐tagged Rab18 mutants, immunoblotted with the indicated antibodies. Rab18WT and Rab18Q67L preferentially associated with kinesin‐1 (KIF5A), whereas Rab18S22N preferentially bound Hsp90α and nSMase2. (B) Schematic summary of Rab18‐GTP– and Rab18‐GDP–specific effector interactions. (C, D) Spinning disk microscopy of NIH3T3 cells co‐expressing Hsp90α‐tagRFP and EGFP‐Rab18 mutants (C), with quantification of cells showing perinuclear Hsp90α accumulation (D). Open arrows, diffuse cytoplasmic distribution of Hsp90α in a Rab18‐GTP‐expressing cell. Scale bar, 20 µm. *** p < 0.001; chi‐square test; n = 88–125 cells. (E, F) Ceramide immunocytochemistry of NIH3T3 cells treated with DMSO, CID (40 µM), or CID plus GW4869 (1.25 µM, 24 h), shown at low and high magnification (E), with quantification (F). Arrows indicate peripheral ceramide accumulation. Scale bars, 20 µm. * p < 0.05; ** p < 0.01; ***p < 0.001; one‐way ANOVA; n = 20.

Article Snippet: EGFP‐Rab18 (Addgene #49550; RRID: Addgene_49550), EGFP‐Rab18Q67L (Addgene #49596; RRID: Addgene_49596), and EGFP‐Rab18S22N (Addgene #49597; RRID: Addgene_49597) were gifts from Marci Scidmore (Huang et al. ).

Techniques: Incubation, Microscopy, Expressing, Immunocytochemistry

Journal: Journal of Extracellular Biology

Article Title: Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

doi: 10.1002/jex2.70112

Figure Lengend Snippet: GDP‐bound Rab18 promotes perinuclear SHH‐XLEV secretion. (A–C) Spinning disk microscopy of NIH3T3 cells coexpressing SHHN‐tagRFP and EGFP‐Rab18 mutants, shown as z‐projections (A), single planes (A), and side views (B), with quantification of perinuclear SHH accumulation (C). Dotted lines, periphery of the Rab18‐GTP‐expressing colony (green) and basolateral SHH deposition (magenta). Arrowheads, colocalization spots of Rab18‐GTP and SHH on the plasma membrane (A) and Rab18‐GTP‐specific basolateral SHH deposition (B). Scale bars, 20 µm. *** p < 0.001; chi‐square test; n = 88–125 cells. Related to . (D) Spinning disk microscopy of NIH3T3 cells undergoing burst‐like secretion of SHHN‐EGFP and Hsp90α‐tagRFP following prolonged CID treatment (arrowheads, 30 h). Scale bar, 20 µm. Related to Figure and . (E, F) Three‐dimensional reconstruction of surface SHH immunofluorescence (red) in SHHN‐EGFP–expressing NIH3T3 cells (E), with quantification of SHH signal intensity appearing on the particle surface (E, arrowheads; F). Scale bar, 20 µm. **** p < 0.0001; Welch's t test; n = 27 versus 90 cells. (G) Working model of the PI3K–Rab18‐GDP–dependent SHH‐XLEV secretion pathway. Related to Figure .

Article Snippet: EGFP‐Rab18 (Addgene #49550; RRID: Addgene_49550), EGFP‐Rab18Q67L (Addgene #49596; RRID: Addgene_49596), and EGFP‐Rab18S22N (Addgene #49597; RRID: Addgene_49597) were gifts from Marci Scidmore (Huang et al. ).

Techniques: Microscopy, Expressing, Clinical Proteomics, Membrane, Immunofluorescence

Journal: International journal of molecular sciences

Article Title: The Rab18/Ras/ERK/FosB/MMP3 Signaling Pathway Mediates Cell Migration Regulation by 2'3'-cGAMP.

doi: 10.3390/ijms26125758

Figure Lengend Snippet: Figure 1. Phospho-profiling identifies MAPK as a critical kinase signaling downstream of 2′3′- cGAMP/Rab18. (A) Immunoblot (IB) analysis of whole-cell lysates (WCL) from the indicated MDA-MB-

Article Snippet: The antibodies used in this study are listed as follows: anti-FOSB2 antibody (Cell Signaling Technology 2251S, Danvers, MA, USA); anti-phospho-ERK1/2 (Thr202/Tyr204) antibody (Cell Signaling Technology 4370S); anti-ERK1 antibody (Santa Cruz Biotechnology sc-271269, Dallas, TX, USA); anti-Rab18 antibody (Santa Cruz Biotechnology sc-393169); anti-Vinculin antibody (Santa Cruz Biotechnology sc-25336); anti-GAPDH antibody (Proteintech 60004-1-Ig, Rosemont, IL, USA); anti-rabbit immunoglobulin G (IgG); horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology 7074); anti-mouse IgG; and HRP-linked antibody (Cell Signaling Technology 7076).

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: The Rab18/Ras/ERK/FosB/MMP3 Signaling Pathway Mediates Cell Migration Regulation by 2'3'-cGAMP.

doi: 10.3390/ijms26125758

Figure Lengend Snippet: Figure 3. MMP3 is a potential transcriptional target of the 2′3′-cGAMP/Rab18/MAPK/FosB2 signaling axis. (A) Representative immunofluorescent (IF) images of indicated MDA-MB-231 cells with DAPI and phalloidin staining. Where indicated, cells were treated with 2.5 µg/mL 2′3′-cGAMP for 24 h. (B) Representative IF images of indicated HeLa cells. Where indicated, HeLa cells were treated with 2.5 µg/mL 2′3′-cGAMP alone, PD-0325901 (1 µM) alone, or in combination for 24 h. (C,D) RT-PCR analysis of mRNA expression of indicated MMPs from MDA-MB-231 cells treated with 2.5 µg/mL 2′3′-cGAMP for either 18 h (C) or 36 h (D). Error bars were calculated as the mean ± SD; n biological replicates are as indicated in each panel. * p < 0.05 (one-way ANOVA test). (E) RT-PCR analysis of mRNA expression of indicated MMPs from MDA-MB-231 cells treated with 2.5 µg/mL 2′3′-cGAMP alone or in combination with SCH772984 (2 µM) for 12 h. Error bars were calculated as the mean ± SD; n biological replicates are as indicated in each panel. * p < 0.05 (one-way ANOVA test). (F) RT-PCR analysis of mRNA expression of indicated MMPs from indicated MDA-MB-231 cells treated with 2.5 µg/mL 2′3′-cGAMP for 18 h. Error bars were calculated as the mean ± SD; n biological replicates are as indicated in each panel. * p < 0.05 (one-way ANOVA test).

Article Snippet: The antibodies used in this study are listed as follows: anti-FOSB2 antibody (Cell Signaling Technology 2251S, Danvers, MA, USA); anti-phospho-ERK1/2 (Thr202/Tyr204) antibody (Cell Signaling Technology 4370S); anti-ERK1 antibody (Santa Cruz Biotechnology sc-271269, Dallas, TX, USA); anti-Rab18 antibody (Santa Cruz Biotechnology sc-393169); anti-Vinculin antibody (Santa Cruz Biotechnology sc-25336); anti-GAPDH antibody (Proteintech 60004-1-Ig, Rosemont, IL, USA); anti-rabbit immunoglobulin G (IgG); horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology 7074); anti-mouse IgG; and HRP-linked antibody (Cell Signaling Technology 7076).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing