Journal: Oncogenesis

Article Title: Sec62 promotes early recurrence of hepatocellular carcinoma through activating integrinα/CAV1 signalling

doi: 10.1038/s41389-019-0183-6

Figure Lengend Snippet: a Left: Functional pathway analysis of the differentially expressed genes was conducted using commercially available IPA software. The change in integrin signalling in both Huh7-Sec62-shRNA cells and Huh7-Sec62-overexpressing cells is consistent with the expression of Sec62. The colour indicates the degree of down- (blue) or up- (orange) regulation following Sec62 knockdown or overexpression in Huh7 cells. Upper right: Cellular movement regulated by Sec62 was identified in both Huh7-Sec62-RNAi cells and Huh7-Sec62-overexpressing cells by GO functional analysis. Lower right: Interaction of Sec62 and integrin signalling after Sec 62 knockdown in Huh7 cells, which was determined based on the integrin signalling interactome in the Ingenuity IPA database overlaid with microarray data from Huh7-Sec62-RNAi cells. b Left: Integrin α5 and integrin αV were overexpressed in Sec62-knockdown (Sec62 − ) Huh 7 cells, and the healing ability of these cells was evaluated by a monolayer wounding assay. Western blot analysis of integrin α 5 and integrin αV expression in Sec62-knockdown cells. Middle: Transwell assay analysis of the migration ability of Sec62 − / integrin α5 (top) or Sec62 − / integrin αV (low) cells. The number of cells that invaded through the filter into the lower compartment was determined using a colorimetric crystal violet assay. Data are presented as the means ± SD of at least three independent experiments and were compared to the amounts of invaded cells from negative control transfected cells. * P < 0.01 versus the control group (Student’s t test). Right: Western blot of co-immunoprecipitated integrin αV (top) and integrin α5 (low) in Huh7 cells after Co-IP with an anti-Sec62 antibody. c The levels of integrin αV, integrin α 5, CAV1, calpains and MLCK expression in the integrin α/CAV1 pathway from HCC patient samples with recurrence ( n = 4) or without recurrence ( n = 4) were evaluated by Western blot analysis.

Article Snippet: Rabbit anti-integrin α2b (#13807), integrin α4 (#8440), integrin α5 (#98204), integrin αV (#60896), CAV1 (#3267), phospho-SHC (Tyr317, #2431), phospho-SHC (Tyr239/240, #2434), SHC (#2432), SOS1 (#5890), Bcar3 (#24032), Calpain 1 (#2556), Calpain 2 (#2539), antibodies were procured from Cell Signal Technology.

Techniques: Functional Assay, Software, shRNA, Expressing, Over Expression, Microarray, Western Blot, Transwell Assay, Migration, Crystal Violet Assay, Negative Control, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Oncogenesis

Article Title: Sec62 promotes early recurrence of hepatocellular carcinoma through activating integrinα/CAV1 signalling

doi: 10.1038/s41389-019-0183-6

Figure Lengend Snippet: Luciferase-labelled Huh7 cells with or without stable Sec62 knockdown were subcutaneously injected into the right axillary, and then, the xenografts were orthotopically implanted into the livers of nude mice. Mice underwent HCC resection on day 14 after implantation. a Left: tumour recurrences were detected after tumour resection with the IVIS system. Then, luciferase-labelled Huh7 cells with or without stable Sec62 overexpression were subcutaneously injected into the right axillary, and then, the xenografts were orthotopically implanted into the livers of nude mice. Mice underwent HCC resection on day 10 after implantation. a Right: tumour recurrences were detected after tumour resection with the IVIS system. b Quantitative fluorescence data in mice. * P < 0.01 versus the control group (Student’s t test). c Western blot analysis of Sec62, integrin αV, integrin α 5, CAV1, calpains and MLCK expression in the resected tumour is shown.

Article Snippet: Rabbit anti-integrin α2b (#13807), integrin α4 (#8440), integrin α5 (#98204), integrin αV (#60896), CAV1 (#3267), phospho-SHC (Tyr317, #2431), phospho-SHC (Tyr239/240, #2434), SHC (#2432), SOS1 (#5890), Bcar3 (#24032), Calpain 1 (#2556), Calpain 2 (#2539), antibodies were procured from Cell Signal Technology.

Techniques: Luciferase, Injection, Over Expression, Fluorescence, Western Blot, Expressing

Journal: Materials Today Bio

Article Title: A macrophage-like biomimetic nanoparticle with high-efficiency biofilm disruption and innate immunity activation for implant-related infection therapy

doi: 10.1016/j.mtbio.2025.101575

Figure Lengend Snippet: Antibiofilm properties of F/R@PM. ( A ) The photographs of S. aureus biofilm processed with different treatments and subjected to crystal violet staining. ( B ) Biofilm biomass of S. aureus biofilm after various treatments. (n = 3, ∗∗ p < 0.01). ( C ) Detached bacteria biomass from S. aureus biofilm after various treatments. (n = 3, ∗∗ p < 0.01). ( D ) Representative SEM images of S. aureus biofilm. ( E ) Representative fluorescence images of S. aureus biofilm stained with SYTO 9/PI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The membranes were previously treated with primary antibodies against α4 (anti-integrin α4, 8440S, CST), β1 (anti-integrin β1, 34971, CST), and CD47 (anti-CD47 antibody, ab175388, Abcam).

Techniques: Staining, Bacteria, Fluorescence

Journal: Nature Communications

Article Title: In vivo self-assembled siRNA as a modality for combination therapy of ulcerative colitis

doi: 10.1038/s41467-022-33436-0

Figure Lengend Snippet: a The CMV-siR TNF-α circuit contains a CMV promoter part and a TNF-α siRNA-expressing part. When the CMV-siR TNF-α circuit is taken up and processed by the liver after intravenous injection, the CMV promoter drives the transcription of TNF-α siRNA in cytoplasm, which leads to the loading of saturated TNF-α siRNA into small extracellular vesicles (sEVs) as cargo. Subsequently, TNF-α siRNA-encapsulating sEVs facilitate the systematic distribution of siRNAs to multiple tissues and cells, including colonic macrophages. Once arriving colonic macrophages, TNF-α siRNA has the potential to regulate immune balance and relieve intestinal inflammation by inhibiting the uncontrolled release of TNF-α from macrophages. b The CMV-siR T+B+I circuit contains a CMV promoter part and a multiple siRNA-expressing part carrying three siRNA expression cassettes organised as a head-to-tail tandem array to simultaneously drive the transcription of TNF-α siRNA, B7-1 siRNA and integrin α4 siRNA. B7-1 siRNA is expected to target antigen-presenting cells (APCs) and inhibit the costimulatory signal to T cells. Integrin α4 siRNA is expected to target α4β7 + CD4 + T cells and block T cell homing to sites of inflammation. Created with BioRender.com.

Article Snippet: They were then incubated with TNF-α (Abcam, ab183218, Cambridge, UK), B7-1 (Abcam, ab254579, Cambridge, UK), integrin α4 (Cell Signalling Technology, 8440S, Danvers, MA, USA), F4/80 (Abcam, ab60343, Cambridge, UK) or CD4 (Proteintech, CL488-65141) antibodies and then washed and incubated with goat anti-rabbit IgG (H + L) and CoraLite594-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00013-4, Wuhan, China).

Techniques: Expressing, Injection, Blocking Assay

Journal: Nature Communications

Article Title: In vivo self-assembled siRNA as a modality for combination therapy of ulcerative colitis

doi: 10.1038/s41467-022-33436-0

Figure Lengend Snippet: a Kinetics of TNF-α siRNA in the mouse colon, spleen and kidney following tail vein injection of the 5 mg/kg CMV-siR TNF-α circuit ( n = 3 in each group). The total TNF-α siRNA levels in the colon, spleen and kidney were calculated based on the total amount of RNA isolated from one gram of each tissue and the TNF-α siRNA content in one μg of total RNA. b In situ detection of TNF-α siRNA in colon sections of DSS mice at 0, 6, 12, 24 or 48 h after injection with the CMV-siR TNF-α circuit. Positive in situ hybridisation signals are shown in green, and DAPI-stained nuclei are shown in blue. Scale bar: 50 μm. c Kinetics of TNF-α siRNA in monocytes and macrophages derived from peripheral blood, spleen and colonic lamina propria of DSS mice following tail vein injection of the 5 mg/kg CMV-siR TNF-α circuit ( n = 3 in each group). d Kinetics of TNF-α siRNA in CD4 + T cells derived from peripheral blood, spleen and colonic lamina propria of DSS mice following tail vein injection of the 5 mg/kg CMV-siR TNF-α circuit ( n = 3 in each group).

Article Snippet: They were then incubated with TNF-α (Abcam, ab183218, Cambridge, UK), B7-1 (Abcam, ab254579, Cambridge, UK), integrin α4 (Cell Signalling Technology, 8440S, Danvers, MA, USA), F4/80 (Abcam, ab60343, Cambridge, UK) or CD4 (Proteintech, CL488-65141) antibodies and then washed and incubated with goat anti-rabbit IgG (H + L) and CoraLite594-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00013-4, Wuhan, China).

Techniques: Injection, Isolation, In Situ, Hybridization, Staining, Derivative Assay

Journal: Nature Communications

Article Title: In vivo self-assembled siRNA as a modality for combination therapy of ulcerative colitis

doi: 10.1038/s41467-022-33436-0

Figure Lengend Snippet: a Flow chart of the experimental design. b Body weight curves ( n = 5 in each group). c Digital Acceleration Index (DAI) scores ( n = 5 in each group). d Representative macroscopic features of colons. Scale bar: 1 cm. e Quantitative RT-PCR analysis of the relative expression levels of TNF-α mRNA in the colon ( n = 5 in each group). f Determination of the absolute expression levels of TNF-α protein in the colon by ELISA ( n = 5 in each group). g Representative images of H&E staining of colon sections. Scale bar: 100 μm. Each experiment was repeated independently three times, and representative results are shown. h , i Quantitative RT-PCR analysis of the relative expression levels of CD206 and NOS2 mRNA in the colon ( n = 6 in each group). j Representative flow cytometric characterisation of colonic macrophages (CD11b + CD64 + ) subdivided into M1-like (Ly6C high CD206 low ) and M2-like (Ly6C low CD206 high ) macrophages. k Bar graph depicting the ratio between M2-like versus M1-like macrophages ( n = 5 in each group). l , n Lamina propria mononuclear cells (LPMCs) were stimulated with PMA/Ionomycin mixture and BFA/Monensin mixture for 5 h, and the percentage of CD4 + IFN-γ + cells and CD4 + IL-17A + cells in CD4 + T cells was measured by flow cytometry. m , o Bar graph depicting the percentage of CD4 + IFN-γ + T cells ( n = 3 in CMV-scrR group; n = 6 in CMV-siR TNF-α group) and CD4 + IL-17A + T cells ( n = 4 in CMV-scrR group; n = 6 in CMV-siR TNF-α group). Values are presented as the mean ± SEM. Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison in ( c , e , f , h , i , k , m , o ) or using two-way ANOVA followed by Dunnett’s multiple comparison in panel b. * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: They were then incubated with TNF-α (Abcam, ab183218, Cambridge, UK), B7-1 (Abcam, ab254579, Cambridge, UK), integrin α4 (Cell Signalling Technology, 8440S, Danvers, MA, USA), F4/80 (Abcam, ab60343, Cambridge, UK) or CD4 (Proteintech, CL488-65141) antibodies and then washed and incubated with goat anti-rabbit IgG (H + L) and CoraLite594-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00013-4, Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Comparison

Journal: Nature Communications

Article Title: In vivo self-assembled siRNA as a modality for combination therapy of ulcerative colitis

doi: 10.1038/s41467-022-33436-0

Figure Lengend Snippet: a Quantitative RT-PCR analysis of the relative expression levels of TNF-α mRNA in the colon ( n = 6 in each group). b Determination of the absolute expression levels of TNF-α protein in the colon by ELISA ( n = 6 in each group). c Quantitative RT-PCR analysis of the relative expression levels of B7-1 mRNA in the colon ( n = 6 in each group). d Representative flow cytometric plots of B7-1 on the surface of colonic lamina propria mononuclear cells. IgG isotype-labelled cells was used as a negative control. e The population of B7-1 + cells in total colonic lamina propria mononuclear cells ( LPMCs) ( n = 4 in each group). f Immunofluorescence staining of B7-1 (red), F4/80 (green) and DAPI (blue) in colon sections. Double-positive (red and green) signals indicate B7-1 + macrophages. Scale bar: 100 μm. Each immunofluorescence staining was repeated independently three times, and representative images are shown. g Quantitative RT-PCR analysis of the relative expression levels of integrin α4 mRNA in the colon ( n = 6 in each group). h Representative flow cytometric plots of integrin α4 on the surface of mononuclear cells derived from the colonic lamina propria. IgG isotype-labelled cells was used as a negative control. i The population of integrin α4 + lymphocytes in total colonic lamina propria lymphocytes ( n = 4 in each group). j Immunofluorescence staining of integrin α4 (red), CD4 (green) and DAPI (blue) in colon sections. Double-positive (red and green) signals indicate integrin α4 + CD4 + cells. Scale bar: 100 μm. Values are presented as the mean ± SEM. Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison in ( a – c , e , g , i ). * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: They were then incubated with TNF-α (Abcam, ab183218, Cambridge, UK), B7-1 (Abcam, ab254579, Cambridge, UK), integrin α4 (Cell Signalling Technology, 8440S, Danvers, MA, USA), F4/80 (Abcam, ab60343, Cambridge, UK) or CD4 (Proteintech, CL488-65141) antibodies and then washed and incubated with goat anti-rabbit IgG (H + L) and CoraLite594-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00013-4, Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Negative Control, Immunofluorescence, Staining, Derivative Assay, Comparison

Journal: Oncogene

Article Title: LASP-1 – A nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex

doi: 10.1038/onc.2015.166

Figure Lengend Snippet: A) Stable knock down of LASP-1. LASP-1 was stably knocked down by employing short hairpin microRNAs through lentiviral mediated transduction into different breast cancer cells. The knock down efficiency was followed by Western blotting of total cell lysates for LASP-1. β-tubulin served as the loading control. B) MCF7 and MDA-231S cells that were stably silenced for LASP-1 expression were cultured in Matrigel and their growth under 3D conditions was assessed. The micrographs were obtained at 200X magnification. C) Up regulated expression of several cell adhesion genes upon knock down of LASP-1. 20 μg of total cell lysates from non-silenced (NS) and LASP-1 knock down MCF7 cells were separated by SDS-PAGE and blotted for E-cadherin, EpCAM, α4-integrin and MARVELD2. β-tubulin and β-actin served as the loading controls. The band intensities of proteins were quantified by using Image J analysis and normalized to β-actin. The fold change was given above the bands. D) Up regulated e xpression of E-cadherin and p120-catenin occurs upon knock down of LASP-1 in luminal breast cancer cell lines : 20 μg of total cell lysates from non-silenced (NS) and LASP-1 knock down MCF7 and MDA-MB-361 cells were separated by SDS-PAGE and blotted for E-cadherin, p120-catenin and β-catenin. The % of knock down for LASP-1 is given above the LASP-1 bands. The fold change is given above the E-cadherin, p120-catenin and β-catenin bands. The experiment was repeated twice and the representative blot is shown. β-tubulin and β-actin served as the loading controls.

Article Snippet: The antibodies were obtained commercially from various sources – 1) Cell Signaling and Technology -Lamin A/C ( #4777); EpCAM (#2626S); DNMT1 (#5032P); HDAC1 (#5356P); G9a (3306S); Snail1 (3879P); α4-Integrin (#8440S); β-actin (#4970S); Di, Tri-methyl histone H3 (Lys9) (#5327) 2) Thermoscientific – UHRF1 (PA5-27969) 3) BD Biosciences – E-cadherin (610181); p120-catenin (610133); β-catenin (610154) 4) Bethyl laboratories – MARVELD2 (A301-505A) and 5) Sigma/Aldrich – β-tubulin (T0198).

Techniques: Knockdown, Stable Transfection, Transduction, Western Blot, Control, Expressing, Cell Culture, SDS Page