shrab27a  (Addgene inc)

Journal: Cancer Biology & Therapy

Article Title: Challenges and caveats in manipulating extracellular vesicle secretion from pancreatic cancer cells

doi: 10.1080/15384047.2025.2569946

Figure Lengend Snippet: Pooled shRab27a does not KD Rab27a. (a) Schematic of EV production through the action of Rab27a. (b) DNA validation of the full Tet-On shRab27a lentiviral construct following transduction. (c) Growth of KPC cells treated with increasing concentrations of dox. (d) Relative Rab27a expression normalized to Gapdh in KPC PalmGRET tCD19 shRab27a cells with siRab27a or dox treatment (+ = 0.6 µg/mL, ++ = 3 µg/mL. (e) Immunoblotting of shRab27a cells after 48 and 72 h of siRNA or 3 µg/mL dox treatment. (f) Relative Rab27a expression normalized to Gapdh in 5 clones with and without 0.3 µg/mL dox treatment. (g) Immunoblotting of shRab27a clone 4 with and without 3 µg/mL dox at 48 and 72 h probed for Rab27a and α-tubulin. (h) Nanoparticle tracking analysis of EVs isolated from shRab27a clone 4 with and without 3 µg/mL dox. p -values were calculated using an unpaired two-tailed Student's t -test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Cell lines were developed to express the tetracycline-inducible shRab27a for mouse Rab27a (pLKO-Tet-On-shRab27a, Addgene #120930).

Techniques: Biomarker Discovery, Construct, Transduction, Expressing, Western Blot, Clone Assay, Isolation, Two Tailed Test

Journal: Cancer Biology & Therapy

Article Title: Challenges and caveats in manipulating extracellular vesicle secretion from pancreatic cancer cells

doi: 10.1080/15384047.2025.2569946

Figure Lengend Snippet: Rab27a and Rab35 KO do not impact KPC tumor growth. (a) Schematic of the involvement of Rab27a and Rab35 in EV secretion. (b) Immunoblot validation of Rab27a and Rab35 KO probed for Rab27a, Rab35, and α-tubulin. (c) Particle concentration of EVs derived from WT, Rab27a KO, and Rab35 KO cells. (d) Growth of WT, Rab27a KO, and Rab35 KO cells. (e) Tumor volume, weight, and immunofluorescence staining for Ki-67 in KPC WT, Rab27a KO, and Rab35 KO 14-d orthotopic pancreatic tumors implanted into C57BL6 mice. p -values were calculated using an ordinary one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Cell lines were developed to express the tetracycline-inducible shRab27a for mouse Rab27a (pLKO-Tet-On-shRab27a, Addgene #120930).

Techniques: Western Blot, Biomarker Discovery, Concentration Assay, Derivative Assay, Immunofluorescence, Staining

Journal: Cancer Biology & Therapy

Article Title: Challenges and caveats in manipulating extracellular vesicle secretion from pancreatic cancer cells

doi: 10.1080/15384047.2025.2569946

Figure Lengend Snippet: Pooled shRab27a does not KD Rab27a. (a) Schematic of EV production through the action of Rab27a. (b) DNA validation of the full Tet-On shRab27a lentiviral construct following transduction. (c) Growth of KPC cells treated with increasing concentrations of dox. (d) Relative Rab27a expression normalized to Gapdh in KPC PalmGRET tCD19 shRab27a cells with siRab27a or dox treatment (+ = 0.6 µg/mL, ++ = 3 µg/mL. (e) Immunoblotting of shRab27a cells after 48 and 72 h of siRNA or 3 µg/mL dox treatment. (f) Relative Rab27a expression normalized to Gapdh in 5 clones with and without 0.3 µg/mL dox treatment. (g) Immunoblotting of shRab27a clone 4 with and without 3 µg/mL dox at 48 and 72 h probed for Rab27a and α-tubulin. (h) Nanoparticle tracking analysis of EVs isolated from shRab27a clone 4 with and without 3 µg/mL dox. p -values were calculated using an unpaired two-tailed Student's t -test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Cell lines were developed to express the tetracycline-inducible shRab27a for mouse Rab27a (pLKO-Tet-On-shRab27a, Addgene #120930).

Techniques: Biomarker Discovery, Construct, Transduction, Expressing, Western Blot, Clone Assay, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts

doi: 10.1038/s41467-018-05077-9

Figure Lengend Snippet: Mitochondrial components segregate into MVBs and are secreted in exosomes. a Confocal co-localization analysis in HEK293 cells treated with FCCP (4 h) and stained with SSBP1 (red), EEA1 and CD63 (green). Bar, 10 µm. Graph shows means for Mander’s co-localization coefficient for SSBP1 and EEA1 or CD63 ( n = 3). t -test: *** P -value < 0.0001. b Confocal co-localization analysis in HEK293 cells co-transfected with Rab7-Q67L-GFP (Rab7mutGFP, green) and mitoDsRed (red), and immunostained for TFAM (purple) and nuclei (HOECHST 58, blue). Center and right images show high magnification of left images (Rab7-Q67L-GFP and mitoDsRed in upper panels; Rab7-Q67L-GFP and TFAM in lower panels). Charts: Fluorescence profiles along the corresponding white lines. Bar, 10 µm. c Mitochondrial components in the exosome fraction obtained from equal numbers of shControl and shnSMase2 J77 T cells. ATAD3, TFAM, and CD63 were detected by immunoblot; mtDNA was detected by PCR for HVRII . Graph: Quantification of exosomal proteins and mtDNA in a representative experiment ( n = 3). d Flow cytometry analysis of mitochondrial mass (Mitotracker green), intracellular ROS levels (DCFDA staining), oxidized DNA (8-OHdG Ab staining), and endogenous TFAM in HEK293 cells knocked down for nSMase2 and Rab27a. Graphs: Quantification from 5–8 independent experiments (Mean). t -test * P -value < 0.05; ** P -value < 0.001. e Electron microscopy images show defects in mitochondrial ultrastructure and cristae organization in shnSMase2 and shRab27a HEK293 cells. Graph: Quantification of mitochondrial cristae width (Mean). t -test: *** P -value < 0.0001. f Graph: Basal oxygen consumption rate (OCR) of control, shnSMase2 and shRab27a HEK293 cells. Dots represent mean from three independent experiments run in duplicate or triplicate. t -test, * P -value < 0.05, *** P -value < 0.0001. Chart: OCR from shControl, shnSMase2 and shRab27a HEK293 cells in response to oligomycin (Oligo), fccp, and rotenone plus antimycin A (Rot/AA). ( n = 2; mean ± S.E.M.). g Western blot analysis of exosomes from Jurkat T cells left untreated, serum-starved overnight or treated with bafilomycin A. Membranes were blotted for TFAM, CD81, and CD63. Graph: Nanoparticle concentration in the exosomal fractions (mean, two independent experiments). h Western blot analysis of exosomes obtained from Jurkat T cells transfected with control or LC3 siRNA. Graph: Nanoparticle concentrations in the exosomal fractions (mean, n = 2). Western blots are representative out of three independent experiments

Article Snippet: The following plasmids were used: mitoDsRed and mitoYFP, (kindly provided by Prof. L. Scorrano, Venetian Institute for Molecular Medicine, Italy), TFAM-GFP and TFAM-DsRED (generated by inserting the human TFAM cDNA into the pEGFP and DsRED plasmids), EGFP-Rab7A Q67L, LC3-GFP and ATG5-GFP (obtained from Addgene: plasmids 28049, 24920 and 22952, respectively), shRNA pLKO control plasmid, shnSMase2 and shRab27a shRNAs (obtained from Open Biosystems), and CD63-GFP .

Techniques: Staining, Transfection, Fluorescence, Western Blot, Flow Cytometry, Electron Microscopy, Control, Concentration Assay