Journal: Turkish journal of biology = Turk biyoloji dergisi

Article Title: Correction of Griscelli Syndrome Type 2 causing mutations in the RAB27A gene with CRISPR/Cas9.

doi: 10.55730/1300-0152.2705

Figure Lengend Snippet: Figure 2. RAB27A expression by donor and GS-2 MSCs and iPSCs. Donor and GS-2 patient-derived MSCs (IK and YF) were stained for RAB27A expression using flowcytometry (upper left) and immunofluorescent staining of adherent cells in culture (upper right, DAPI: blue, RAB27A: red). RAB27A gene expression in GS-2 MSCs from two donors (İK and YF) was calculated relative to RAB27A expression in healthy donor MSCs, whereas GS-2 iPSCs expression of RAB27A was calculated relative to healthy donor iPSCs (lower left). GS-2 iPSC expression of pluripotency genes was confirmed for OCT4, SOX2, and NANOG (lower right).

Article Snippet: Membranes were stained with an anti-RAB27A antibody (#STJ25258, St John’s Laboratory Ltd, London, UK) at 1:1000 overnight at 4 °C and counterstained with secondary antirabbit-HRP antibody at 1:10000 (#11-4220-82, Invitrogen, Waltham, MA, USA) for 1 h at room temperature.

Techniques: Expressing, Derivative Assay, Staining, Gene Expression

Journal: Turkish journal of biology = Turk biyoloji dergisi

Article Title: Correction of Griscelli Syndrome Type 2 causing mutations in the RAB27A gene with CRISPR/Cas9.

doi: 10.55730/1300-0152.2705

Figure Lengend Snippet: Figure 5. Transfection of GS-2 iPSCs with the RAB27A exon 3 mutation (upper left) resulted in loss of viability and spontaneous differentiation (lower left) and low HDR efficacy (right).

Article Snippet: Membranes were stained with an anti-RAB27A antibody (#STJ25258, St John’s Laboratory Ltd, London, UK) at 1:1000 overnight at 4 °C and counterstained with secondary antirabbit-HRP antibody at 1:10000 (#11-4220-82, Invitrogen, Waltham, MA, USA) for 1 h at room temperature.

Techniques: Transfection, Mutagenesis

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: FLS pathology was accompanied by increased EVP secretion in OA. (A) Gene ontology (GO) cellular component enrichment analysis of differentially expressed genes between normal and OA synovium. (B, C) Violin plots for the expression levels of the exosome secretion marker (RAB27A), senescence markers (CDKN1A, CDKN2A) and inflammation‐related markers (PTGS2, IL6, MMP13), along with Pearson correlation analyses between the expression levels of these markers and RAB27A. Data was normalized as fragments per kilobase of exon model per million mapped fragments (FPKM). (D–G) Multiplex immunohistochemical (mIHC) staining of normal and OA synovium, with magnified views of the boxed areas showing individual colour channels. Scale bar = 100 µm. Line graphs display the relative intensity and co‐localization of each fluorescence along the line in the magnified images. (H, I) Representative RAB27A fluorescence staining images and quantification in the synovial region of sham‐operated mice and mice at 4 and 8 weeks post‐DMM surgery. Scale bar = 25 µm. (J) Schematic diagram of in vitro EVP collection. Briefly, FLSs were pre‐treated with IL‐1β (10 ng/mL) and Bleomycin (25 µg/mL) for 24 h and changed into fresh medium without inducers. EVPs were then isolated from the conditioned medium through ultracentrifugation. (K, L*) Representative MMP13 fluorescence staining and SA‐β‐gal staining images for FLSs after inflammation and senescence induction. (M) Diameter distribution of EVPs from control and pathological FLSs by NTA, with screenshots of the particle flow. (N, O) Representative TEM images for EVPs and Western blot gels for EVP protein markers. Scale bar = 200 µm. (P, Q) Quantification and protein concentration of EVPs isolated from different culture medium, both of which were normalized to original cell counts. ** Indicates p < 0.01, * indicates p < 0.05, ns indicates p > 0.05, versus the indicated groups, one‐way ANOVA.

Article Snippet: The primary antibodies utilized were validated by manufacturers or previously publications: Vimentin (#201158) from zen‐bioscience, MMP13 (18165‐1‐AP) from Proteintech, P16 (ab211542) from Abcam, MMP3 (A11418), iNOS (A3774) and COL2A1 (A1560, IF) from ABclonal, IL‐1β (bs‐25615R) from Bioss, COL2A1 (BA0533) from BOSTER (IHC), RAB27A (DF6702) and SOX9 (AF6330) from Affinity Biosciences.

Techniques: Expressing, Marker, Multiplex Assay, Immunohistochemical staining, Staining, Fluorescence, In Vitro, Isolation, Control, Western Blot, Protein Concentration

Journal: Journal of Extracellular Vesicles

Article Title: Targeted Blockage of Pathological Extracellular Vesicles and Particles From Fibroblast‐Like Synoviocytes for Osteoarthritis Relief: Proteomic Analysis and Cellular Effect

doi: 10.1002/jev2.70162

Figure Lengend Snippet: Intra‐articular injection of FLS‐targeting AAV for delivering Rab27a ‐shRNA to specifically reduce EVP secretion. (A) Rab27a expression levels in FLSs after transfection with control shRNA and Rab27a knockdown shRNA. (B, C) EVP size distribution and quantification after transfection with control shRNA and Rab27a knockdown shRNA. ** Indicates p < 0.01, versus the indicated groups, student’s t ‐test. (D) Representative Western blot images for positive and negative surface markers of EVPs at equal concentration after shRNA transfection. (E) Schematic diagram of constructing a virus targeting FLS to inhibit Rab27a for intra‐articular injection. The synovium‐affinity peptide HAP‐1 is fused with the AAV9 viral capsid viral protein 2 (VP2), and the AAV9 vector is designed to simultaneously carry the gene encoding the mScarlet fluorescent protein and an expression cassette for shRNA. (F) Representative images of mScarlet fluorescence in the synovium and cartilage regions of mouse knee joints. Scale bar = 200 µm and 100 µm separately. (G) Representative images of mScarlet fluorescence in sections of multiple mouse organs. Scale bar = 200 µm. (H) Representative images and quantitative results of RAB27A expression in the synovial region after intra‐articular AAV injection. ** Indicates p < 0.01, versus the indicated groups, two‐way ANOVA.

Article Snippet: The primary antibodies utilized were validated by manufacturers or previously publications: Vimentin (#201158) from zen‐bioscience, MMP13 (18165‐1‐AP) from Proteintech, P16 (ab211542) from Abcam, MMP3 (A11418), iNOS (A3774) and COL2A1 (A1560, IF) from ABclonal, IL‐1β (bs‐25615R) from Bioss, COL2A1 (BA0533) from BOSTER (IHC), RAB27A (DF6702) and SOX9 (AF6330) from Affinity Biosciences.

Techniques: Injection, shRNA, Expressing, Transfection, Control, Knockdown, Western Blot, Concentration Assay, Virus, Plasmid Preparation, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Targeted blockade of extracellular vesicles-mediated profibrotic vicious circle using EVs-based dual-drug delivery system to prevent liver fibrosis

doi: 10.1186/s12951-025-03940-8

Figure Lengend Snippet: Elevated EV secretion in injured-hepatocytes at early stages of liver fibrosis. (A) Schematic illustration of EVs extraction from human peripheral blood. (B) Representative TEM images of plasma-derived EVs from HC and LC (scale bar = 100 nm). (C) Particle and protein concentrations of EVs from healthy controls (HC, n = 13) and patients with liver cirrhosis (LC, n = 18). (D) The gene expression levels of Rab27a, Rab27b in human liver tissues (HC = 7, LC = 11). (E) Schematic illustration of EVs extraction from liver tissues ( n = 3). (F) Representative TEM images of EVs derived from liver tissue (scale bar = 100 nm). (G) Western blot analysis of LT-EVs markers. (H) Particle and protein concentrations of LT-EVs from mice with fibrosis at different weeks. (I) Schematic illustration of the fibrotic mice model. (J) Western blot analysis of Rab27a in liver tissues ( n = 2). (K) Representative images and quantitative analysis of immunohistochemical staining of Rab27a in liver tissues (scale bar = 100 μm, n = 6), and enlarged the boxed areas (scale bar = 50 μm). (L) Schematic illustration of EVs extraction from AML12 cells. (M) Particle and protein concentrations of Ctrl-EVs and CCl 4 -EVs ( n = 3). (N) Representative TEM images of Ctrl-EVs and CCl 4 -EVs (scale bar = 200 nm). (O) Western blot analysis of Ctrl-EVs and CCl 4 -EVs markers. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p ≤ 0.0001

Article Snippet: For immunohistochemistry and immunofluorescence, liver tissues were incubated with Rab27a (HuaBio, China), α-SMA (HuaBio, China), F4/80 (Servicebio, China), CK18 (HuaBio, China) and CD63 (Servicebio, China) with primary antibodies at 4 °C overnight.

Techniques: Extraction, Clinical Proteomics, Derivative Assay, Gene Expression, Western Blot, Immunohistochemical staining, Staining