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: iMeta

Article Title: Dissecting the angiogenic mechanism of pachychoroid polypoidal choroidal vasculopathy

doi: 10.1002/imt2.70176

Figure Lengend Snippet: Effects of OSS on mCECs following Edn1 knockdown and re‐expression and Ednra knockdown. (A) Representative immunofluorescence images of F‐actin and VE‐cadherin. F‐actin length and VE‐cadherin fluorescence intensity were quantified using ImageJ (version 1.54p). n = 3 independent biological replicates per group. (B) Potential targets for single‐cell sequencing based on qRT‐PCR screening. n = 3 independent biological replicates per group. (C) Representative trajectories of mCECs under laminar flows shear stress and oscillatory flow shear stress with and without Edn1 shRNA. The change in displacement in 24 h was determined for each experiment and used as a single data point ( n = 3 independent biological replicates per group). (D) CCK8‐based proliferation and transwell‐based invasion assays assessing the effects of OSS and Edn1 knockdown. Edn1 silencing significantly attenuated the OSS‐induced reduction in proliferation and increase in invasion of mCECs. n = 5 independent biological replicates. (E) Tube formation of mCECs under OSS with or without Edn1 knockdown. OSS promoted tube formation, whereas Edn1 knockdown attenuated this effect. The number of master junctions and total branching length were quantified using ImageJ, n = 3 independent biological replicates. (F) Representative Western blots showing the expression of EDN1, c‐Casp3, ANG‐1, MMP9, IL‐6, and ZO‐1 in response to OSS with and without Edn1 shRNA. n = 3 independent biological replicates. (G) CCK8 assays of mCEC proliferation under OSS following Edn1 re‐expression after Edn1 knockdown. n = 5 independent biological replicates. Transwell invasion (H) and scratch migration (I) assays showing that the OSS‐induced invasive and migratory responses of mCEC cells were attenuated by Edn1 knockdown and restored by Edn1 re‐expression. n = 5 independent biological replicates per group for the Transwell invasion assay and n = 3 for the scratch migration assay. (J) Representative fluorescence images of ROS production in mCECs exposed to OSS following Edn1 knockdown and/or re‐expression. Intracellular ROS levels were assessed by DCF fluorescence. n = 3 independent biological replicates per group. (K) Expression of EDN1, c‐Casp3, ANG‐1, MMP9, IL‐6, and ZO‐1 following Edn1 knockdown and subsequent Edn1 re‐expression. n = 3 independent biological replicates per group. (L) Co‐immunoprecipitation showing the interaction between EDN1 and EDNRA in mCECs protein extracts. n = 3 independent biological replicates. (M) Representative trajectories of mCECs exposed to OSS with or without Ednra knockdown. The change in displacement over 24 h was calculated for each independent experiment. n = 3 independent biological replicates per group. (N) CCK8 assay assessing the effects of OSS and Ednra knockdown on mCEC proliferation. Ednra silencing significantly attenuated the OSS‐induced reduction in proliferation. n = 5 independent biological replicates per group. Scratch migration (O) and Transwell invasion (P) assays showing that OSS increased the migratory and invasive capacities of mCECs and that these effects were attenuated by Ednra knockdown. n = 3 independent biological experiments for the scratch migration assay and n = 5 for the transwell invasion assay. (Q) Ednra silencing reduced the expression of EDNRA, c‐Casp3, ANG‐1, MMP9, and IL‐6 and increased ZO‐1 expression. n = 3 independent biological replicates per group. OSS, oscillatory shear stress (±6 dyn/cm 2 , 12 h); CN, control (laminar shear stress at 6 dyn/cm 2 ); shEdn1, Edn1 shRNA, oeEdn1, over‐expressed Edn1 ; shEdnra, Ednra shRNA; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; CCK‐8, Cell Counting Kit‐8; DCF, dichlorofluorescein; IP, immunoprecipitation; IgG, immunoglobulin G; ROS, reactive oxygen species. Data are presented as mean ± SD from n independent biological replicates. For two‐group comparisons, statistical significance was assessed by two‐tailed unpaired Student's t ‐test; for multiple groups, by one‐way ANOVA followed by Tukey's multiple comparisons test. Independent biological replicates: separate cell‐culture experiments per condition. The single and double asterisks indicate p < 0.05 and p < 0.01, respectively.

Article Snippet: Primary antibodies against EDN1 (Abcam, Cat. No. ab117757), EDNRA (Abcam, Cat. No. ab117521), MMP9 (Santa Cruz, Cat. No. sc‐393859), IL‐6 (HUABIO, Hangzhou, HA601051 ), ZO‐1 (Cell Signaling, Cat. No.13663), c‐Casp3 (Cell Signaling, Cat. No.9661), angiopoietin 1 (HUABIO, Hangzhou, Cat. No. ET1611‐28), and β‐actin (Sigma, Cat. No. A5441) were used.

Techniques: Knockdown, Expressing, Immunofluorescence, Fluorescence, Single-Cell Sequencing, Quantitative RT-PCR, Screening, Shear, shRNA, Western Blot, Migration, Transwell Invasion Assay, Co-Immunoprecipitation Assay, CCK-8 Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Cell Counting, Immunoprecipitation, Two Tailed Test, Cell Culture