Journal: PLOS Neglected Tropical Diseases

Article Title: Immune-mediated remodelling of tissue architecture in Post-Kala-Azar Dermal Leishmaniasis (PKDL)

doi: 10.1371/journal.pntd.0014798

Figure Lengend Snippet: A: Heat map indicating the fold change increase in levels of circulatory levels of cytokine milieu and MMP9 in patients with PKDL (n = 16) with respect to healthy controls (n = 9). The colour bar represents the range of fold change (1-13), with dark blue representing the highest fold change and white being the baseline. B: Representative immunofluorescence profiles of TNF-α expression from dermal biopsies of a healthy control, patient with polymorphic or macular PKDL (20x and 40x magnification), along with bar scatter plots showing the expression of TNF-α in healthy controls (●, n = 6), patients with PKDL (■, n = 16), either polymorphic (▲, n = 8) or macular (▼, n = 8) PKDL; each horizontal bar represents the median (IQR) fluorescence positivity/field. C: Representative immunofluorescence profiles of IFN-γ expression from dermal biopsies of a healthy control, patient with polymorphic and macular PKDL (20x and 40x magnification) along with bar scatter plots indicating the expression of IFN-γ in healthy controls (●, n = 6), patients with PKDL (■, n = 15), either polymorphic (▲, n = 7) or macular (▼, n = 8) PKDL; each horizontal bar represents the median (IQR) fluorescence positivity/field. D: Representative immunohistochemical profiles of MMP9 in dermal biopsies of a healthy control, patient with polymorphic or macular PKDL (10x and 40x magnification). Bar scatter plot demonstrating the status of MMP9 in healthy controls (●, n = 6), patients with PKDL (■, n = 20), either polymorphic (▲, n = 8) or macular (▼, n = 12) PKDL; each horizontal bar represents the median (IQR), along with correlation of MMP9 with lesional TNF-α (n = 14), IFN-γ (n = 12), E-Cadherin (n = 12), fibronectin (n = 12) and laminin (n = 10) fluorescence positivity/field.

Article Snippet: All reagents were obtained from Sigma Aldrich (St. Louis, MO, USA) except anti-human E-Cadherin (clone NCH-38), CD8 (clone C6/144B), CD68 (clone PG-M1) [Dako, CA, USA], CD20 (clone L26), MMP 9 (clone EP127) [BioSB, Santa Barbara, CA, USA], anti-human ICAM-1, TNF-α, IFN-γ [BD Biosciences, San Jose, CA, USA], Fibronectin (FN1, A12392), Lamininβ1 (LAMB1, A9827), Collagen IV (Col4A3, A16359), Collagen I (Col1A1, A1352) and Goat anti-rabbit secondary antibody IgG-FITC (AS011) [Abclonal, Woburn, USA], antifadent [Southern Biotech, Birmingham, AL, USA], EnVision FLEX Target Retrieval Solution [Dako, CA, USA] and secondary detection system [DAB + chromogen, ScyTek Laboratories, West Logan, UT, USA], MMP9 Human ELISA Kit (RayBiotech, GA, USA), Bio-Plex Pro Human Chemokine Panel 40-Plex and 48-plex (for IFN-γ and TNF-α, BioRad, CA, USA).

Techniques: Immunofluorescence, Expressing, Control, Fluorescence, Immunohistochemical staining

Journal: Clinical and Translational Medicine

Article Title: Non‐canonical function of transferrin receptor‐1 promotes breast cancer metastasis by activating HCK‒STAT3‒MMP9 signalling

doi: 10.1002/ctm2.70731

Figure Lengend Snippet: Matrix metalloproteinase 9 (MMP9) is a central downstream executor in transferrin receptor 1 (TfR‐1)‐promoted breast cancer cells mobility. (A) Differentially expressed genes between mock and TfR‐1‐overexpressed MDA‐MB‐231/4175 cells were analysed by Gene Ontology (GO) enrichment. (B) Heatmap depicting gene expression in positive regulation of cell migration pathways for mock versus TfR‐1‐overexpressing MDA‐MB‐231/4175 cells. (C) Core genes in migration‐related pathways were evaluated using degree, betweenness and closeness algorithms. (D) Pearson correlation analysis revealed strong positive correlation between TFRC and MMP9 expression ( R = .991, p < .001). (E) Expression of MMP9 was detected by immunohistochemical (IHC) in 74 non‐metastatic and 66 metastatic breast cancer tissue samples. (F) The expression of MMP9 in the breast cancer tissue microarray was quantified based on staining intensity and extent. (G) Correlation analysis was performed to evaluate the relationship between the expression levels of TfR‐1 and MMP9 in breast cancer tissues. (H) Relative mRNA expression levels of MMP9 in MDA‐MB‐231 and MDA‐MB‐231/4175 mock or TfR‐1‐overexpressing cells were determined by real‐time quantitative PCR (RT‐qPCR). (I) Western blot (WB) analysis of total STAT3, pSTAT3 and MMP9 in MDA‐MB‐231 and MDA‐MB‐231/4175 mock or TfR‐1‐overexpressing cells. (J) MMP9 secretion in culture supernatants was measured by enzyme‐linked immunosorbent assay (ELISA) in MDA‐MB‐231 and MDA‐MB‐231/4175 mock or TfR‐1‐overexpressing cells. (K) ELISA analysis of MMP9 secretion in supernatants from TfR‐1‐overexpressing MDA‐MB‐231 and MDA‐MB‐231/4175 cells treated with or without OKT9. (L) RT‐qPCR analysis of MMP9 mRNA in TfR‐1‐overexpressing cells treated with or without STAT3 inhibitor BP‐1‐102. (M) WB analysis of MMP9, pSTAT3, STAT3 and TfR‐1 in MDA‐MB‐231 and MDA‐MB‐231/4175 TfR‐1‐overexpressing cells with or without BP‐1‐102. (N) ELISA measurement of MMP9 secretion in TfR‐1‐overexpressing MDA‐MB‐231 and MDA‐MB‐231/4175 cells treated with or without BP‐1‐102. Data represent mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was determined by unpaired, two‐tailed Student's t ‐test, * p < .05, ** p < .01, *** p < .001, with comparisons made to relevant controls in each experiment.

Article Snippet: MMP9 in cell culture supernatants was measured using the MMP9 enzyme‐linked immunosorbent assay (ELISA) kit (Abclonal, #RK00217).

Techniques: Gene Expression, Migration, Expressing, Immunohistochemical staining, Microarray, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: Non‐canonical function of transferrin receptor‐1 promotes breast cancer metastasis by activating HCK‒STAT3‒MMP9 signalling

doi: 10.1002/ctm2.70731

Figure Lengend Snippet: STAT3‒matrix metalloproteinase 9 (MMP9) signalling mediates the pro‐metastatic effects of transferrin receptor 1 (TfR‐1) in breast cancer. (A) Western blot (WB) analysis of TfR‐1 and MMP9 expression in MMP9 depletion breast cancer cells. (B) Enzyme‐linked immunosorbent assay (ELISA) analysis of secreted MMP9 levels after siMMP9 transfection. (C) Transwell assays comparing siNC‐ or siMMP9‐treated cells. (D) ELISA analysis of secreted MMP9 in mock versus TfR‐1‐overexpressing breast cancer cells transfected with siNC or siMMP9. (E) Transwell assays of mock versus TfR‐1‐overexpressing cells transfected with siNC or siMMP9. (F) Effects of MMP9 knockdown and STAT3 inhibition (BP‐1‐102, 10 µM, 24 h) on MMP9 secretion. (G) Transwell assays of siNC‐ and siMMP9‐treated cells with or without STAT3 inhibitor BP‐1‐102 (10 µM, 24 h). Data are shown as mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was determined by unpaired, two‐tailed Student's t ‐test, * p < .05, ** p < .01, *** p < .001, with comparisons made to relevant controls in each experiment. (H) Schematic summary of TfR‐1‐induced haematopoietic cell kinase (HCK) axis activation promoting breast cancer metastasis.

Article Snippet: MMP9 in cell culture supernatants was measured using the MMP9 enzyme‐linked immunosorbent assay (ELISA) kit (Abclonal, #RK00217).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown, Inhibition, Standard Deviation, Two Tailed Test, Activation Assay