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: NPJ Regenerative Medicine

Article Title: Tubular epithelial cells-derived small extracellular vesicle-VEGF-A promotes peritubular capillary repair in ischemic kidney injury

doi: 10.1038/s41536-022-00268-x

Figure Lengend Snippet: a Schematic diagram of the experimental design. Briefly, mice were subjected to a 28-min bilateral IRI at day 7-post lentiviral shRab27a injection, and were euthanized at 3 days after reperfusion. Schematic created with BioRender.com. b Effects of Rab27a knockdown on the serum creatinine after IRI ( n = 5). c Representative images of PAS-stained kidneys (top) or Rab27a-immunostained kidney sections (bottom). Scale bars, 50 μm. d Quantification of kidney injury based on PAS staining ( n = 5). e RT-qPCR analysis of KIM-1 mRNA levels in kidneys ( n = 5). f Quantification of Rab27a + tubules based on five mice, with at least ten sections counted in each. g Western blotting analysis of PCNA and Rab27a expression in kidneys ( n = 5). h Representative confocal images of CD31- and PCNA-stained kidney sections. Scale bars, 50 μm. Quantification of PTC intensity and CD31 + PCNA + cells (white arrowheads) based on five mice, with at least ten sections counted in each. Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. NC group. shRab27a, lentivirus containing shRNA against Rab27a. NC, negative control lentivirus. Two-tailed Student’s t test.

Article Snippet: To inhibit sEV secretion in the kidney, Rab27a was knocked down by the administration of 5 × 10 7 TU of lentivirus carrying short hairpin RNA against Rab27a (shRab27a) (5′-GATGCACGCGTACTGTGAA-3′) or negative control (NC) (5′-TTCTCCGAACGTGTCACGT-3′) (GeneChem) via tail vein injection one week before I/R surgery.

Techniques: Injection, Knockdown, Staining, Quantitative RT-PCR, Western Blot, Expressing, shRNA, Negative Control, Two Tailed Test