Journal: Nature Communications

Article Title: Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90

doi: 10.1038/s41467-017-00726-x

Figure Lengend Snippet: Tumor cell-released Hsp70/90-expressing EVs induce muscle catabolism in myotubes. a CD9-positive EVs induce catabolic response in C2C12 myotubes. EVs isolated from LCM were subjected to immunoprecipitation (IP) against CD9 with pre-immune IgG as control. Resulted pellet and supernatant (Sup) were used to treat C2C12 myotubes and compared with PBS and input EVs for catabolic activity using western blotting. Data ( n = 3) were analyzed by analysis of variance (ANOVA) and * denotes a difference from PBS treatment ( P < 0.05). b Hsp70/90 release by cachexia-inducing tumor cells is dependent on EV release. Rab27a and Rab27b were knocked down by transfecting LLC and C26 cells with control or specific siRNAs. EVs and Hsp70/90 released into conditioned media were quantified after Brij98 treatment. EVs were quantified by AchE. Data ( n = 3) were analyzed by Student t -test. c Tumor cell-induced catabolic response in myotubes is dependent on EV release. C2C12 myotubes were treated with conditioned media of Rab27-deficient LLC or C26 cells and analyzed for p38 MAPK activity (at 1 h), atrogin1 (at 8 h) and MHC levels (72 h). Conditioned medium of non-tumorigenic NL20 cells (NCM) was used as control. Data ( n = 3) were analyzed by ANOVA. * denotes a difference ( P < 0.05) from NCM-treated control. # denotes a difference between bracketed columns

Article Snippet: Antibodies for UBR2 (#NBP1-45243), LC3 (#NB100-2220), Rab27A (#SC-74586), Rab27B (#NBP1-79631) were from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Isolation, Immunoprecipitation, Control, Activity Assay, Western Blot

Journal: Nature Communications

Article Title: Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90

doi: 10.1038/s41467-017-00726-x

Figure Lengend Snippet: Tumor cell-released Hsp70/90-expressing EVs are critical to the development of muscle wasting in mice. a Elevation of serum Hsp70/90 in LLC tumor-bearing mice is dependent on EV release. LLC cells transduced with lentivirus encoding Rab27a- and Rab27b-specific shRNA or empty vector were analyzed for knockdown effect by western blotting ( top ). Mice were then implanted with Rab27-deficient or control LLC cells. In 21 days serum levels of EVs, Hsp70/90 were analyzed. b Rab27-deficient LLC tumor does not induce muscle catabolism in mice. Mice derived from a were analyzed for activity of the catabolic markers in TA. c Rab27-deficient LLC tumor does not induce muscle wasting in mice. Mice derived from a were analyzed for muscle wasting. Scale bar , 100 μm. Data ( n = 6) were analyzed by analysis of variance or χ 2 analysis (for CSA). * denotes a difference ( P < 0.05)

Article Snippet: Antibodies for UBR2 (#NBP1-45243), LC3 (#NB100-2220), Rab27A (#SC-74586), Rab27B (#NBP1-79631) were from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Transduction, shRNA, Plasmid Preparation, Knockdown, Western Blot, Control, Derivative Assay, Activity Assay

Journal: The international journal of biochemistry & cell biology

Article Title: Abnormal Expression of Rab27B in Prostatic Epithelial Cells of Benign Prostatic Hyperplasia Alters Intercellular Communication

doi: 10.1016/j.biocel.2020.105898

Figure Lengend Snippet: A. Plasma membrane proteins were isolated from HEK293 cells overexpressing Rab27A, Rab27B or control vector. The levels of the indicated membrane proteins in total cell extracts (Ext) and plasma membrane (PM) fractions were analyzed by western blotting. B. Quantitative presentations of the relative levels of the plasma membrane proteins from the cells shown in A. Data are from three independent experiments and expressed as mean ± SD. * P < 0.01. C. Plasma membrane proteins were isolated from RWPE-1 cells overexpressing Rab27B or control vector. The levels of the indicated membrane proteins in total cell extracts and plasma membrane fractions were analyzed by western blotting. D. Quantitative presentations of the relative levels of the plasma membrane proteins from the cells shown in C. Data are from three independent experiments and expressed as mean ± SD. * P < 0.01. E. The levels of IGF-1 in media conditioned by RWPE-1 expressing Rab27B or control vector. Data are from three independent experiments and expressed as mean ± SD. * P < 0.05.

Article Snippet: Samples were sectioned at 5 μm, and immunostaining was performed with primary Rab27B antibody using the ImmunoCruz rabbit ABC staining System (Santa Cruz Biotechnology, Santa Cruz, CA) followed by Vector NovaRED substrate (Vector Laboratories, Burlingame, CA).

Techniques: Clinical Proteomics, Membrane, Isolation, Control, Plasmid Preparation, Western Blot, Expressing

Journal: The international journal of biochemistry & cell biology

Article Title: Abnormal Expression of Rab27B in Prostatic Epithelial Cells of Benign Prostatic Hyperplasia Alters Intercellular Communication

doi: 10.1016/j.biocel.2020.105898

Figure Lengend Snippet: A. Immunohistochemistry (IHC) images showing the expression of Rab27B in BPH and adjacent normal (N) prostatic tissues. Scale bar = 100 μM. B. Quantitative presentations of the Rab27B expression levels in BPH and normal prostatic tissues (NAP) analyzed by IHC. C. Violin plot showing the centrality analysis of the expression of Rab27B in BPH and healthy (normal) prostates. D. Western blots and quantitative presentations showing the expression levels of Rab27B in prostatic epithelial cells derived from normal prostate (RWPE-1), BPH tissues (BPH-1) and benign prostate tissues (BHPrE-1). Quantitative presentation data are from three independent experiments and expressed as mean ± SD. * P < 0.01.

Article Snippet: Samples were sectioned at 5 μm, and immunostaining was performed with primary Rab27B antibody using the ImmunoCruz rabbit ABC staining System (Santa Cruz Biotechnology, Santa Cruz, CA) followed by Vector NovaRED substrate (Vector Laboratories, Burlingame, CA).

Techniques: Immunohistochemistry, Expressing, Western Blot, Derivative Assay

Journal: The international journal of biochemistry & cell biology

Article Title: Abnormal Expression of Rab27B in Prostatic Epithelial Cells of Benign Prostatic Hyperplasia Alters Intercellular Communication

doi: 10.1016/j.biocel.2020.105898

Figure Lengend Snippet: A. BPH-1 cells stably expressing scrambled control shRNA (ctrl) or Rab27B specific shRNAs were analyzed by western blotting for the levels of Rab27B. The expression level of Rab27B in RWPE-1 was included for comparison. B. The growth rates of BPH-1 cells stably expressing control shRNA (ctrl) or Rab27B specific shRNAs were analyzed. The data are from four independent experiments and the growth differences at 72 hr between the control and the two Rab27B shRNAs expressed as mean ± SD. * P < 0.01. C. The levels of the activation-dependent phosphorylation of ERK1/2, AKT, S6, 4E-BP1 and their expression in cells stably expressing control or Rab27B specific shRNAs were analyzed by western blotting. D. Quantitative presentations of the phosphorylation levels of ERK1/2, AKT and S6 in the cells shown in C. The data were obtained by comparing the ratio of phosphorylated / total protein level of the Rab27 shRNA expressing cells with that of the control cells. Data are from three independent experiments and expressed as mean ± SD. * P < 0.01.

Article Snippet: Samples were sectioned at 5 μm, and immunostaining was performed with primary Rab27B antibody using the ImmunoCruz rabbit ABC staining System (Santa Cruz Biotechnology, Santa Cruz, CA) followed by Vector NovaRED substrate (Vector Laboratories, Burlingame, CA).

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Comparison, Activation Assay, Phospho-proteomics

Journal: The international journal of biochemistry & cell biology

Article Title: Abnormal Expression of Rab27B in Prostatic Epithelial Cells of Benign Prostatic Hyperplasia Alters Intercellular Communication

doi: 10.1016/j.biocel.2020.105898

Figure Lengend Snippet: A. The levels of the indicated proteins in the total cell extracts (Ext) and plasma membrane (PM) of BPH-1 cells expressing control or Rab27B specific shRNAs were analyzed by western blotting. B. Quantitative presentations of the relative levels of the plasma membrane proteins from the cells shown in A. The data are from three independent experiments and expressed as mean ± SD. * P < 0.01. C. Confocal images of IGF-1Rβ distribution in BPH-1 cells expressing control or Rab27B specific shRNAs. IGF-1Rβ was detected with IGF-1Rβ antibody (red), actin fibers with Alexa fluor-488 phalloidin (green) and nuclei (blue) with DAPI. White lines in the merged images mark where the cross-sections of XZ plane were taken. Scale bar = 20 μm. D. The levels of IGF-1 in media conditioned by BPH-1 cells expressing control or Rab27B specific shRNAs. Data are from three independent experiments and expressed as mean ± SD. * P < 0.01. E. The growth curves of WPMY-1 cells in media conditioned by BPH-1 cells expressing control or Rab27B specific shRNAs. Data are from four independent experiments. The growth differences at 72 hr between the cells cultured by conditioned media from the control and the two Rab27B shRNAs knockdown cells are expressed as mean ± SD. * P < 0.01.

Article Snippet: Samples were sectioned at 5 μm, and immunostaining was performed with primary Rab27B antibody using the ImmunoCruz rabbit ABC staining System (Santa Cruz Biotechnology, Santa Cruz, CA) followed by Vector NovaRED substrate (Vector Laboratories, Burlingame, CA).

Techniques: Clinical Proteomics, Membrane, Expressing, Control, Western Blot, Cell Culture, Knockdown

Journal: The international journal of biochemistry & cell biology

Article Title: Abnormal Expression of Rab27B in Prostatic Epithelial Cells of Benign Prostatic Hyperplasia Alters Intercellular Communication

doi: 10.1016/j.biocel.2020.105898

Figure Lengend Snippet: A. Western blotting analysis of activation-dependent phosphorylation of ERK1/2, AKT and S6 in HEK293 cells overexpressing Rab27A, Rab27B or control vector. B. Quantitative presentations of activation-dependent phosphorylation of ERK1/2, AKT and S6 in the cells shown in A. The data were obtained by comparing the ratio of phosphorylated / total levels of the indicated proteins in cells overexpressing Rab27A or Rab27B with that of the cells expressing a control vector. Data are from three independent experiments and expressed as mean ± SD. * P < 0.01. C. Western blotting analysis of activation-dependent phosphorylation of ERK1/2, AKT and S6 in uninfected RWPE-1 cells (ctrl) or those infected with lentiviral particles expressing empty vector or Rab27B. D. Quantitative presentations of activation-dependent phosphorylation of ERK1/2, AKT and S6 in the cells shown in C. Data are from three independent experiments and expressed as mean ± SD. * P < 0.01.

Article Snippet: Samples were sectioned at 5 μm, and immunostaining was performed with primary Rab27B antibody using the ImmunoCruz rabbit ABC staining System (Santa Cruz Biotechnology, Santa Cruz, CA) followed by Vector NovaRED substrate (Vector Laboratories, Burlingame, CA).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Control, Plasmid Preparation, Expressing, Infection

Journal: The Journal of Cell Biology

Article Title: Kinesin-1 controls mast cell degranulation and anaphylaxis through PI3K-dependent recruitment to the granular Slp3/Rab27b complex

doi: 10.1083/jcb.201605073

Figure Lengend Snippet: Characterization of the kinesin-1–dependent transport machinery in MCs. (A) Relative quantification of Rab27a, Rab27b, Slp1, Slp2, Slp3, Slp4, and Slp5 transcripts in BMMCs, using real-time PCRs. Transcript levels for each sample were expressed as a proportion of the mean value for Rab27b. (B) BMMCs were lysed and then analyzed by Western blotting with anti-Kif5b, anti-Slp2, anti-Slp3, and anti-Rab27 antibodies. (C) IgE-sensitized WT BMMCs were either not stimulated (NS) or were stimulated with 20 ng/ml DNP-HSA for 10 min and then lysed. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-Rab27 antibody or with an isotype control antibody. The immunoblots were analyzed using anti-Kif5b, anti-Slp3, and anti-Rab27 antibodies. (D) Flag-Rab27b and GFP-Slp3 were coexpressed in HEK 293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and then separated by SDS-PAGE. Coprecipitated Rab27b and Slp3 were immunoblotted with anti-Flag and anti-GFP. (E) Flag-Rab27b, -Slp3, and GFP-KLC1 were coexpressed in HEK 293T cells. Cells were either not stimulated or were stimulated with PMA/ionomycin, and then cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and separated by SDS-PAGE. Coprecipitated Rab27b or Slp3 and KLC1 were immunoblotted with anti-Flag and anti-GFP. (F) Flag-Rab27b, -Slp3, and GFP-Kif5b were coexpressed into HEK 293T cells. Cells were either not stimulated or were stimulated with PMA/ionomycin, and then cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and separated by SDS-PAGE. Coprecipitated Rab27b or Slp3 and Kif5b were immunoblotted with anti-Flag and anti-GFP antibodies. Data in C–F are representative of three independent experiments. (G) Schematic diagram of the heterotrimeric protein transport complex involved in the translocation of SGs upon activation.

Article Snippet: Levels of KIF5A , KIF5B , KIF5C , KLC1 , KLC2 , KLC3 , RAB27A , RAB27B , SYTL1 , SYTL2 , SYTL3 , SYTL4 , and SYTL5 transcripts were determined in quantitative PCR assays using TaqMan Gene Expression Master Mix primer ( KIF5A : Mm00515265_m1; KIF5B : Mm00515276_m1; KIF5C : Mm00500464_m1; RAB27A : Mm00469997_m1; KLC1 : Mm00492936_m1; KLC2 : Mm00492945_m1; KLC3 : Mm00461422_m1; RAB27B : Mm00472653_m1; SYTL1 : Mm00473300_m1; SYTL2 : Mm01317927_m1; SYTL3 : Mm00473333_m1; SYTL4 : Mm00489110_m1; SYTL5 : Mm00624760_m1; and 18S : Mm03928990_g1; Applied Biosystems) and cDNA.

Techniques: Quantitative Proteomics, Western Blot, Immunoprecipitation, Control, SDS Page, Translocation Assay, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Kinesin-1 controls mast cell degranulation and anaphylaxis through PI3K-dependent recruitment to the granular Slp3/Rab27b complex

doi: 10.1083/jcb.201605073

Figure Lengend Snippet: Subcellular localization of the kinesin-1–dependent transport machinery in MCs. (A–D) Confocal microscopy of WT or Kif5b-deficient BMMCs transfected with GFP-Rab27b and Dsred-Slp3 and labeled with anti-STX3 antibody in resting cells (A and C) or in cells stimulated by the addition of 20 ng/ml DNP-HSA for 10 min (B and D). (E) The statistical analysis of experiments in B and D was performed using an unpaired t test. ***, P < 0.0001. More than 30 cells were counted per setting. (F–J) IgE-sensitized WT (F and H) and cKO Kif5b (G, I, and J) BMMCs were plated on fibronectin-coated glass coverslips and either were not stimulated (F, G, and I) or were stimulated by the addition of 20 ng/ml DNP-HSA for 10 min (H and J). Cells were then fixed, permeabilized, and stained with an anti-tubulin antibody and an anti-Kif5b or anti-STX3 antibody. Bars, 2 µm. All images of single cells are representative of >100 cells observed over at least three independent experiments. (K) Statistical analysis of microtubule reorganization at the plasma membrane identified by filopodia-like extension shape in experiments in H and J was performed using an unpaired t test. More than 100 cells were counted per setting. NS, not stimulated.

Article Snippet: Levels of KIF5A , KIF5B , KIF5C , KLC1 , KLC2 , KLC3 , RAB27A , RAB27B , SYTL1 , SYTL2 , SYTL3 , SYTL4 , and SYTL5 transcripts were determined in quantitative PCR assays using TaqMan Gene Expression Master Mix primer ( KIF5A : Mm00515265_m1; KIF5B : Mm00515276_m1; KIF5C : Mm00500464_m1; RAB27A : Mm00469997_m1; KLC1 : Mm00492936_m1; KLC2 : Mm00492945_m1; KLC3 : Mm00461422_m1; RAB27B : Mm00472653_m1; SYTL1 : Mm00473300_m1; SYTL2 : Mm01317927_m1; SYTL3 : Mm00473333_m1; SYTL4 : Mm00489110_m1; SYTL5 : Mm00624760_m1; and 18S : Mm03928990_g1; Applied Biosystems) and cDNA.

Techniques: Confocal Microscopy, Transfection, Labeling, Staining, Clinical Proteomics, Membrane

Journal: Materials Today Bio

Article Title: pH-responsive 2D niobium carbide nanosheets for targeted circPUM1 siRNA delivery in ovarian cancer therapy

doi: 10.1016/j.mtbio.2025.102314

Figure Lengend Snippet: Scheme 1. General design of P-Nb 2 C-PEG@si-circPUM1 in ovarian cancer. (A) Schematic illustration of P-Nb 2 C-PEG preparation. The precursor (Nb 2 AlC) underwent HF etching followed by exfoliation with TPAOH, yielding negatively charged monolayer Nb 2 C nanosheets. After mixture with excess PEI, P-Nb 2 C was generated with a positive surface charge. We modified the nanosheets with CHO-PEG-CHO, where the aldehyde groups form Schiff base linkages with amine groups, enabling PEG grafting. (B) P-Nb 2 C-PEG was loaded with circPUM1 siRNA via electrostatic interaction and circulated in blood (pH 7.4). Upon reaching the acidic tumor microenvironment (pH 6.5), the nanosheets underwent pH-responsive charge reversal enabling effective cellular uptake. (C) The internalized circPUM1 siRNA specifically targeted the back-splice junction of circPUM1, disrupted its circular structure and abolished its miRNA-sponging activity. The released miRNAs suppressed the expression of VEGFA and RAB27B, thereby inhibiting RAB27B-mediated exosome secretion and VEGFA-triggered angiogenesis. (Created in BioRender. Guan, X. (2025) https://BioRender.com/x7gxaqp )

Article Snippet: Membranes were subsequently blocked with NcmBlot blocking buffer (NCM Biotech, China) for 10 min. Primary antibody incubation was carried out at 4 °C overnight with specific antibodies against VEGFA (1:1000; Boster, China), RAB27B (1:1000; Boster, China), and β-actin (1:8000; Abbkine, China).

Techniques: Generated, Modification, Activity Assay, Expressing

Journal: Materials Today Bio

Article Title: pH-responsive 2D niobium carbide nanosheets for targeted circPUM1 siRNA delivery in ovarian cancer therapy

doi: 10.1016/j.mtbio.2025.102314

Figure Lengend Snippet: CircPUM1 promotes angiogenesis in ovarian cancer as the form of exosomes. (A) The expression of circPUM1 was positively related with MVD in ovarian cancer tissue. (B) Experimental design schematic (Created in BioRender. Guan, X. (2025) https://BioRender.com/f5br0q0 ): HUVEC were co-cultured with exosomes derived from circPUM1-overexpressing and control OVCAR3 cells. (C) RT-qPCR showed that circPUM1 was highly expressed in HUVEC after co-cultured with exosomal circPUM1. Exosomal circPUM1 enhanced migration ability (D, E), viability (F), invasiveness (G), and tube formation ability (H) of HUVECs. (I) Circular RNA pull-down assays using biotinylated circPUM1 probes confirmed co-enrichment of circPUM1 and miR-607. (J) Dual-luciferase reporter assays showed miR-607 targeting 3′-UTR in both VEGFA and RAB27B . (K) Western blot revealed that overexpression of circPUM1 upregulated VEGFA and RAB27B , whereas miR-607 downregulated these targets in OVCAR3 cells. Exosomal circPUM1 elevated intracellular VEGFA expression in HUVECs. (L) Molecular schematic (Created in BioRender. Guan, X. (2025) https://BioRender.com/5eldsz5 ): In ovarian cancer, the highly expressed circPUM1 sponges miR-607, upregulating expression of VEGFA and RAB27B . This dual regulation establishes a pro-tumorigenic cascade through two distinct pathways: (1) RAB27B -mediated secretion of exosomal circPUM1, and (2) VEGFA -induced activation of angiogenic signaling.

Article Snippet: Membranes were subsequently blocked with NcmBlot blocking buffer (NCM Biotech, China) for 10 min. Primary antibody incubation was carried out at 4 °C overnight with specific antibodies against VEGFA (1:1000; Boster, China), RAB27B (1:1000; Boster, China), and β-actin (1:8000; Abbkine, China).

Techniques: Expressing, Cell Culture, Derivative Assay, Control, Quantitative RT-PCR, Migration, Luciferase, Western Blot, Over Expression, Activation Assay

Journal: Materials Today Bio

Article Title: pH-responsive 2D niobium carbide nanosheets for targeted circPUM1 siRNA delivery in ovarian cancer therapy

doi: 10.1016/j.mtbio.2025.102314

Figure Lengend Snippet: In vitro antitumor performance of P-Nb 2 C@si-circPUM1 in ovarian cancer cells. (A) Confocal microscopy images showed intracellular uptake of Cy3-labeled circPUM1 siRNA. Cell apoptosis assays (B), Transwell assays (C), wound healing assays (E) and EdU proliferation assays (F) showed that P-Nb 2 C-loaded circPUM1 siRNA effectively suppressed ovarian cancer cell phenotypes. (G) RT-qPCR demonstrated a significant downregulation of circPUM1 expression in lipo2000-transfected and P-Nb 2 C-loaded siRNA groups. Immunofluorescence (D) and Western blot (H) validated the downregulation of VEGFA and RAB27B expression in cells treated with P-Nb 2 C@si-circPUM1. (I) CCK8 assays showed inhibition of ovarian cancer cell viability in lipo2000-transfected and P-Nb 2 C-loaded siRNA groups.

Article Snippet: Membranes were subsequently blocked with NcmBlot blocking buffer (NCM Biotech, China) for 10 min. Primary antibody incubation was carried out at 4 °C overnight with specific antibodies against VEGFA (1:1000; Boster, China), RAB27B (1:1000; Boster, China), and β-actin (1:8000; Abbkine, China).

Techniques: In Vitro, Confocal Microscopy, Labeling, Quantitative RT-PCR, Expressing, Transfection, Immunofluorescence, Western Blot, Inhibition

Journal: Materials Today Bio

Article Title: pH-responsive 2D niobium carbide nanosheets for targeted circPUM1 siRNA delivery in ovarian cancer therapy

doi: 10.1016/j.mtbio.2025.102314

Figure Lengend Snippet: Molecular mechanism validation of P-Nb 2 C-PEG@si-circPUM1 anti-angiogenic performance in vivo . (A) H&E staining showed increased necrotic loci within tumors of P-Nb 2 C-PEG@si-circPUM1 group. (B, D) IHC staining of Ki-67 demonstrated a clear reduction in Ki-67-positive cells in P-Nb 2 C-PEG@si-circPUM1 group. (C, F) IHC staining of CD31 indicated a marked decrease in MVD within tumors of P-Nb 2 C-PEG@si-circPUM1 group. (H) QPCR showed a significant downregulation of circPUM1 expression in the P-Nb 2 C-PEG@si-circPUM1 treated group. IHC (E, G) and Western blot (I, J) demonstrated significant down-regulation of RAB27B and VEGFA .

Article Snippet: Membranes were subsequently blocked with NcmBlot blocking buffer (NCM Biotech, China) for 10 min. Primary antibody incubation was carried out at 4 °C overnight with specific antibodies against VEGFA (1:1000; Boster, China), RAB27B (1:1000; Boster, China), and β-actin (1:8000; Abbkine, China).

Techniques: Biomarker Discovery, In Vivo, Staining, Immunohistochemistry, Expressing, Western Blot

Journal: Molecular Autism

Article Title: Platelets of mice heterozygous for neurobeachin, a candidate gene for autism spectrum disorder, display protein changes related to aberrant protein kinase A activity

doi: 10.1186/2040-2392-4-43

Figure Lengend Snippet: Normal expression of actin and the proteins involved in dense granule formation and secretion in platelets of Nbea +/- mice. (A) Western blot analysis detected no changes in the total actin levels in platelets of Nbea +/- mice compared to Nbea +/+ mice. Actin expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content and the expression in platelets of wild-type mice was set at 100% (n = 4/genotype). (B) The total amount of Munc13-4, Rab27b and Calmodulin was unaltered in platelets of Nbea +/- mice. Protein levels were normalized to actin and the expression level in platelets of Nbea +/+ mice was set at 100% (n = 4 samples/genotype).

Article Snippet: Proteins were transferred to Protan Nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) and incubated with antibodies against Munc13-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1/1,000), Rab27b (homemade antibody, 1/1,000), Calmodulin (kindly provided by Professor H Desmedt, KU Leuven; 1/1,000), Talin-1 (Cell Signaling Technology, Danvers, MA, USA; 1/500), Calpain-2 large subunit (Cell Signaling Technology; 1/1000), Calpain-4 regulatory subunit (Santa Cruz Biotechnology; 1/1,000), phospho-(Ser/Thr) PKA substrate antibody (Cell Signaling Technology; 1/1,000), and actin (Sigma-Aldrich, St Louis, MO, USA; 1/5,000), used for normalization.

Techniques: Expressing, Western Blot

Journal: NPJ Parkinson's Disease

Article Title: Transcriptomic profiling of early synucleinopathy in rats induced with preformed fibrils

doi: 10.1038/s41531-023-00620-y

Figure Lengend Snippet: a Diagram of the vesicular release process during neurotransmission, showing the docking, priming, fusion pore opening, and fusion stages. Proteins encoded by transcripts downregulated in early synucleinopathy are highlighted in green. b Fischer 344 rats received two intrastriatal injections of 4 µg/µL α-syn PFFs or PBS (Vehicle), SN was collected 2-months after PFF administration from flash-frozen tissue and processed for ddPCR. Graphs show mRNA from genes in the general vesicular release process normalized to Rpl13a mRNA and measured via ddPCR. Transcripts assessed were Rab3a (Ras-associated binding protein 3a), Rab3c (Ras-associated binding protein 3c), Rab27b (Ras-associated binding protein 27b), Vamp2 (synaptobrevin 2), Stx1b (syntaxin 1b), Syt1 (synaptotagmin 1), Syt2 (synaptotagmin 2), Syt3 (synaptotagmin 3), Snap25 (synaptosome associated protein 25), Stxbp1 (syntaxin binding protein 1, also known as MUNC18), Pclo (piccolo), Bsn (bassoon), Erc2 (ELKS/RAB6-interacting/CAST family member 2, also known as CAST1), Rims1 (regulating synaptic membrane exocytosis 1), Cplx1 (complexin 1), Cplx2 (complexin 2), and Nsf (N-ethylmaleimide sensitive factor, also known as vesicular fusion protein NSF). Columns indicate the group means, circles represent individual data points ( n = 8 per group before outlier removal), and error bars represent ±1 standard error of the mean. An asterisk represents significance ( p ≤ 0.05). Outliers were removed based on the absolute deviation from the median method. c Male Fischer 344 rats received two intrastriatal injections of 4 µg/µL α-syn PFFs, brains were removed 2-months after PFF administration and processed for fluorescent in situ hybridization (FISH). Panels show representative Syt1 FISH micrographs from the SNpc White box denotes the area of the higher magnification images, which show Syt1 mRNA (red) and immunofluorescent staining for pSyn (green). White arrows denote neurons containing pSyn inclusions which show a qualitative decrease in Syt1 mRNA. Scale bars are 500 µm in the lower magnification image, and 10 µm in the higher magnification images.

Article Snippet: Probes used for rats were Aldh1a1 (Applied Biosystems # Rn00755484_m1, Lot# P230119-000 E06, FAM-MGB), Bsn (Applied Biosystems # Rn00569626_m1, Lot# 2041626, FAM-MGB), Cplx1 (Applied Biosystems # Rn02396766_m1, Lot# 1545259, FAM-MGB), Cplx2 (Applied Biosystems # Rn01441919_g1, Lot# 1823042, FAM-MGB), Ddc (Applied Biosystems # Rn01401189_m1, Lot# 2021653, FAM-MGB), Drd2 (Applied Biosystems # Rn00561126_m1, Lot# 1970620, FAM-MGB), Erc2 (Applied Biosystems # Rn00596745_m1, Lot# P230401-000 B05, FAM-MGB), Nsf (Applied Biosystems # Rn00572694_m1, Lot# P230401-000 B06, FAM-MGB), Pclo (Applied Biosystems # Rn00571796_m1, Lot# Rn00571796, FAM-MGB), Rab3a (Applied Biosystems # Rn00564615_m1, Lot# P230401-000 B01, FAM-MGB), Rab3c (Applied Biosystems # Rn01493822_m1, Lot# P230401-000 B02, FAM-MGB), Rab27b (Applied Biosystems # Rn00584945_m1, Lot# P230401-000 B07, FAM-MGB), Rgs8 (Applied Biosystems # Rn00571066_m1, Lot# 1616092, FAM-MGB), Slc6a3 (Applied Biosystems # Rn00562224_m1, Lot# 1771212, FAM-MGB), Slc18a2 (Applied Biosystems # Rn00564688_m1, Lot# 1728686, FAM-MGB), Snap25 (Applied Biosystems # Rn00578534_m1, Lot# 1830682, FAM-MGB), Snca (Applied Biosystems # Rn00569821_m1, Lot# 1765929, FAM-MGB), Sncg (Applied Biosystems # Rn00581652_m1, Lot# 1764532, FAM-MGB), Stxbp1 (Applied Biosystems # Rn00564767_m1, Lot# P230401-000 B03, FAM-MGB), Stx1b (Applied Biosystems # Rn01510167_m1, Lot# P230401-000 B04, FAM-MGB), Syn1 (Applied Biosystems # Rn00569468_m1, Lot# 1695933, FAM-MGB), Syn2 (Applied Biosystems # Rn00569739_m1, Lot# 1416478, FAM-MGB), Syn3 (Applied Biosystems # Rn00567380_m1, Lot# 1581173, FAM-MGB), Syt1 (Applied Biosystems # Rn00436862_m1, Lot# 1564760, FAM-MGB), Syt2 (Applied Biosystems # Rn00561994_m1, Lot# 1821869, FAM-MGB), Syt3 (Applied Biosystems # Rn00569396_m1, Lot# 1821502, FAM-MGB), Th (Applied Biosystems # Rn00562500_m1, Lot# 2013779, FAM-MGB), Vamp2 (Applied Biosystems # Rn01465442_m1, Lot# 1403518, FAM-MGB) and the reference probe was Rpl13a (Applied Biosystems # Rn00821946_g1, Lot# P230401-004 H11, VIC-MGB).

Techniques: Binding Assay, Membrane, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A) Boxplot derived from gene expression data in GEPIA comparing Rab27A and Rab27B expression in normal tissue and colon adenocarcinoma (COAD) tissue (TCGA database online website GEPIA: http://gepia.cancer-pku.cn/ , Normal = 349, Tumor = 275.). (B) Analysis of Rab27A and Rab27B expression in normal tissue (n=3) and CRC cell lines (DLD1, SW480, HCA7, HCT116, HT29, and MOSER) by RT-PCR. All data presented are the average ± SEM of three independent experimental repeats. (C) Immunohistochemistry images of staged colon adenocarcinoma and adjacent normal tissue from Biomax tissue microarray stained with Rab27B. Best representative images are presented. (D) Plot showing immunoreactivity score (IRS) (n=90). (E) WB showing the comparison of Rab27B expression in normal tissues vs. CRC cell lines (SW480, HCA7, HCT116, Caco-2, HT29, and DLD1). Actin was used as the loading control. (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: Derivative Assay, Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Microarray, Staining, Comparison, Control

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A-D) Depletion of Rab27B by siRNA-mediated knockdown resulted in the formation of vesicle-like bodies (indicated by red arrows) in HCT116 (A, B) and SW480 cells (C, D) . (E) Rab27B was knocked out by CRISPR/Cas9 in HCT116 cells, resulting in vesicle formation in the KO cells (F) . Knockdown and knockout were verified by WB using actin as the loading control. Brightfield images were taken to observe cell morphology. All data presented are representative of four independent experiments. (G) Electron microscopy images of HCT116 WT and Rab27B KO cells. Rab27B KO cells showed an accumulation of autophagosome-like vesicles. Images are representatives of two independent experiments.

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: Knockdown, CRISPR, Knock-Out, Control, Electron Microscopy

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A) Individual Rab27B KO clones were isolated and screened by WB for the loss of Rab27B protein. Actin was used as loading control. (B) RT-PCR was performed to measure Rab27A expression in Rab27B KO cells.

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: Clone Assay, Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A) Immunofluorescnece images of HCT116 WT and Rab27B KO stained with CYTO-ID dye to label autophagic vesicles and Dapi to stain the nucleus. WT cells treated with rapamycin (0.5 um) and chloroquine (10 um) overnight were used as positive control. (B) Comparison of CYTO-ID positive vesicles/cell in WT and KO cells. (C) Absorbance (499/548 nm) for CYTO-ID was measured. (D) HCT116 and Rab27B KO cells were immune stained with anti-α-tubulin (red signal) and anti-LC3 (green signal) antibody. Higher magnification images of the area outlined in white show the presence of punctate LC3-II positive structures in Rab27B KO cells (white arrows). Dapi (blue) was used to label the nucleus. (E) siRNA-mediated knockdown of Rab27B in CRC cell line SW480 was followed by immunofluorescence staining. Number of LC3 positive vesicles per cell was scored in HCT116 Rab27B KO (H) and siRab27B SW480 cells (I) . (F) Rab27B KO cells were transfected with pcDNA3.1zeo(+) vector containing full-length human Rab27B (Gene Universal Inc.). Transfected cells were selected in media containing Zeocin (500 ug/ml), and stable addback clones were selected and expanded. (F, J) Immunofluorescence staining and scoring for LC3 showed rescue of autophagy phenotype in the addback cells. (G) Immunofluorescence staining of HCT116 control, siRab27A, and siRab27B cells with anti-α-tubulin (red signal) and anti-LC3 (green signal) antibodies . (K) Rab27A KD did not result in autophagic vesicle accumulation, as seen in Rab27B KD cells. Data presented are representative of three independent experiments (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: Staining, Positive Control, Comparison, Knockdown, Immunofluorescence, Transfection, Plasmid Preparation, Clone Assay, Control

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A) HCT116 and Rab27B KO cell lysates were probed for LC3-II by western blot analysis. Rab27B KO cells showed increased LC3-II compared to HCT116. Cell lysates were also probed for Rab27B to verify the absence of Rab27B in the KO cells. Actin was used as the loading control. (C, E) WB showing LC3 level in Rab27B KD HCT116 and SW480 cells. (B, D, F) Densitometry analysis of LC3-II/LC3-I ratio using ImageJ software. LC3-II/LC3-I was increased in Rab27B depleted cells. (G) Western blot of HCT116 wt, Rab27B KO, and Rab27B addback cells shows restoration of LC3-II level in addback cells.

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: Western Blot, Control, Software

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A) Heatmap of RNA seq transcriptome analysis of HCT116 WT and Rab27B KO cells. The scale at the right indicates relative expression levels from high (red) to low (blue). (B) Comparison of mRNA expression of different autophagy genes (ULK1, ULK2, FIP200, ATG14, BECLIN1, VPS34, ATG7, ATG3, ATG5, ATG12, STX17, SNAP29, VAMP8, and GABARAP) between WT and KO cells obtained from RNA seq analysis. (C) IF staining of HCT116 and Rab27B KO cells with autophagy marker p62 (green) and Dapi (blue). The KO cells exhibited increased oveall p62 fluorescence intensity (D) and a higher percentage of cells with p62 positive vesicles (E) . (F) Lysotracker staining of HCT116 and Rab27B KO cells to label autolysosomes. Dapi was used for nuclear staining. Cells kept in DMEM without glucose (12h) were used as positive controls. Rab27B KO cells showed reduced lysotracker-positive vesicles (indicated by white arrows), suggesting impairment in lysosomal fusion. (G) Rab27B was depleted by siRNA-mediated KD in HCT116 cells stably expressing mCherry-EGFP-LC3B. Autophagy flux was monitored by comparing the ratio of mCherry and GFP double-positive vesicles (yellow) and mCherry-positive (red) vesicles. Cells treated with rapamycin (0.5um for 12h) were used as positive controls. Data presented are representative of three independent experiments (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: RNA Sequencing, Expressing, Comparison, Staining, Marker, Fluorescence, Stable Transfection

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: Rab27B KO cells were transfected with EGFP-Rab27B fusion constructs (Constitutively active Q78L mutant, dominant negative N133I and T23N mutants, and Geralyngeralynation mutant GER) as well as wt Rab27B (A) . Stable cell lines expressing the mutant proteins were established and confirmed by WB analysis for Rab27B (B) . (C) Brightfield images to observe the morphology and autophagic vesicle accumulation in mutant Rab27B cell lines show only wt and constitutively active (Q78L) mutant Rab27B reversed autophagy dysregulation. The dominant negative (N133I and T23N) or GER mutants failed to restore functional autophagy. (D, E) MTT assays were performed to assess the cell viability of mutant Rab27B cell lines over time. HCT116 wt and Rab27B KO cells were used as controls. Cells expressing constitutively active (Q78L) mutant Rab27B showed a higher proliferation rate than WT or KO cells (D), whereas no change was observed in the dominant negative (T23N) mutant cell line (E) . Three independent assays were performed.

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: Transfection, Construct, Mutagenesis, Dominant Negative Mutation, Stable Transfection, Expressing, Functional Assay

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A) Brightfield images of spheroids formed by HCT116 WT and Rab27B KO cells at day 4, day 14 and day 21. (B) . The area of the spheroids was measured and calculated using ImageJ software. Rab27B KO cells show significant impairment in spheroid growth. (C, D) Rab27B deletion significantly reduces colony formation in soft agar colony formation assay. The results shown are representative of three independent experiments. (E, F) Rab27B deletion inhibits xenograft tumor growth in vivo . HCT116 wt and two separate Rab27B KO clones were injected (100 ul, 1 million cells) into the flanks of 5-6 weeks athymic nude mice (n=8 tumors /group). Tumors were measured thrice weekly. The Mice were sacrificed, and tumors were excised, weighed, measured, and photographed next to a ruler 28 days after injection. (E) Representative images of mice and harvested tumors from control and Rab27B KO groups. (F) Tumor volume were calculated by the formula- [length x (width) 2 /2]. Values given are expressed as mean ± SEM (n=8 per group). (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: Software, Soft Agar Assay, In Vivo, Clone Assay, Injection, Control

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: (A) HCT116 and KO cells were grown in 96-well plates containing DMEM with 10% FBS for 48 hours, followed by serum starvation in only DMEM (4.5 g/l glucose), DMEM with low glucose (1g/l glucose) and DMEM without any glucose for 24 hours. (B) Cell survival was measured by MTT assay normalized to the initial OD value before starvation. (C) Western blotting shows an increase in Rab27B protein level following starvation. Actin was used as the loading control. Experiments were repeated three times. The results are shown as mean ± SEM. (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Article Snippet: Rab27B was knocked out in the human colon cancer cell line HCT116 using Rab27B CRISPR/Cas9 KO plasmid (sc-403498; Santa Cruz Biotechnology), followed by insertion of a homology-directed repair (HDR) plasmid (sc-4403498-HDR Santa Cruz Biotechnology) into the double-stranded break.

Techniques: MTT Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

doi: 10.1101/2024.03.22.586334

Figure Lengend Snippet: Rab27B KO cells were transfected with EGFP-Rab27B fusion constructs (Constitutively active Q78L mutant, dominant negative N133I and T23N mutants, and Geralyngeralynation mutant GER) as well as wt Rab27B (A) . Stable cell lines expressing the mutant proteins were established and confirmed by WB analysis for Rab27B (B) . (C) Brightfield images to observe the morphology and autophagic vesicle accumulation in mutant Rab27B cell lines show only wt and constitutively active (Q78L) mutant Rab27B reversed autophagy dysregulation. The dominant negative (N133I and T23N) or GER mutants failed to restore functional autophagy. (D, E) MTT assays were performed to assess the cell viability of mutant Rab27B cell lines over time. HCT116 wt and Rab27B KO cells were used as controls. Cells expressing constitutively active (Q78L) mutant Rab27B showed a higher proliferation rate than WT or KO cells (D), whereas no change was observed in the dominant negative (T23N) mutant cell line (E) . Three independent assays were performed.

Article Snippet: Stable clones were screened using western blotting and RT PCR. peGFP-C1 vectors containing full-length Rab27B (GFP-Rab27B, Addgene plasmid #89447) and mutant forms of Rab27B that encoded constitutively active Q78L (GFP-Rab27B Q78L, Addgene plasmid #89448) dominant negative T23N (GFP-Rab27B T23N, Addgene plasmid #89450), dominant negative N133I (GFP-Rab27B N133I, Adddgene plasmid #89449, and geranylgeranyl-binding mutant (GER) (GFP-Rab27B GG, Addgene plasmid #89451) were gifted by Wendy Westbroek (Reference).

Techniques: Transfection, Construct, Mutagenesis, Dominant Negative Mutation, Stable Transfection, Expressing, Functional Assay