Journal: bioRxiv

Article Title: High-content screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia

doi: 10.1101/2020.02.28.959189

Figure Lengend Snippet: (A) Brightfield microscopy showing a representative example of nasal epithelial cells in BEGM culture (PCD 10) at passage 1. (B) Representative example of brightfield microscopy showing nasal epithelial cells (PCD 10) in 3T3+Y culture at passage 2. Scale bar = 100µm. Part of this image previously appeared in a schematic diagram in a published methods chapter . Reprinted by permission from Springer Nature: Springer eBook, Methods in Molecular Biology, Springer Science Business Media New York (2019). (C) Cumulative population doublings for four healthy donor nasal epithelial cell cultures in BEGM (grey) and 3T3+Y (black). (D) Cumulative population doublings for three primary ciliary dyskinesia (PCD) patient nasal epithelial cells in BEGM (grey) and 3T3+Y (black). (E) Representative examples of immunofluorescence images showing pan-keratin, keratin 5 and P63 expression (red) in three PCD patient 3T3+Y cultures. The nuclear counterstain DAPI is shown (blue). Scale bar = 100 µm. (F-J) qPCR analysis comparing the expression of keratin 5 ( KRT5 ; F), KRT14 (G), TP63 (H), integrin alpha 6 ( ITGA6 ; I) and nerve growth factor receptor ( NGFR ; J) between healthy and PCD donor basal cell cultures in 3T3+Y. P values were derived using two-tailed paired t-tests, ns indicates P > 0.05.

Article Snippet: The following Taqman pre-designed, inventoried probes, along with 2x PCR Master Mix (Applied Biosciences), were used for qPCR reactions: KRT5 (Hs00361185_m1), KRT14 (Hs00265033_m1), KRT8 (Hs01595539_g1), TP63 (Hs00978339_m1), NGFR (Hs00609977_m1), ITGA6 (Hs01041011_m1), MUC5AC (Hs01365616_m1), MUC5B (Hs00861595_m1), FOXJ1 (Hs00230964_m1) and B2M (Hs00187842_m1). qPCR was performed under standard conditions using an Eppendorf Real-Time PCR machine.

Techniques: Microscopy, Immunofluorescence, Expressing, Derivative Assay, Two Tailed Test

Journal: Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society

Article Title: RNA sequencing data from neutrophils of patients with cystic fibrosis reveals potential for developing biomarkers for pulmonary exacerbations

doi: 10.1016/j.jcf.2018.05.014

Figure Lengend Snippet: Top 10 down-regulated and top 10 up-regulated differentially expressed genes in neutrophils from CF patients before and after therapy

Article Snippet: The assays used in this study were ADCY3 (Hs01086502_m1), ANXA3 (Hs00974395_m1), ATP9A (Hs00391058_m1), AZU1 (Hs00156049_m1), CD177 (Hs00360669_m1), CD44 (Hs01075861_m1), CST7 (Hs00175361_m1), CTSG (Hs00175195_m1), DEFA4 (Hs00157252_m1), ELANE (Hs00236952_m1), GRINA (Hs00418129_g1), ITGA7 (Hs01056475_m1), LCN2 (Hs01008571_m1), MPEG1 (Hs00909102_s1), MPO (Hs00165162_m1), OLFM4 (Hs00197437_m1), PFKFB3 (Hs00998700_m1), PRTN3 (Hs00160521_m1), S100A12 (Hs00942835_g1) with 18s (Hs99999901_s1) as reference gene (Applied Biosystems).

Techniques:

Journal: Angiogenesis

Article Title: TFEB controls integrin-mediated endothelial cell adhesion by the regulation of cholesterol metabolism

doi: 10.1007/s10456-022-09840-x

Figure Lengend Snippet: TFEB silencing increases the integrin protein levels. A qPCR of ITGB1, ITGB3, ITGA5 and ITGAV expression in scr-shRNA and sh-TFEB-ECs. Data are expressed as relative fold change in sh-TFEB-ECs compared with the expression in scr-shRNA-ECs after normalization to the housekeeping gene TBP ( n = 3 independent experiments, values as mean ± SEM; p = ns sh-TFEB-ECs versus scr-shRNA-ECs by Student’s t test). B Representative western blot of active and total β1 integrins immunoprecipitated from scr-shRNA- or sh-TFEB-ECs lysates. After immunoprecipitation (IP) with anti-active β1 integrin (9EG7) and anti-total β1 integrin antibodies, proteins were blotted (IB) with anti-total β1 integrin antibody. In the same lysates used for immunoprecipitation, the endogenous expression of total β1 integrin and β-tubulin were blotted with the specific antibodies and presented as “input”. C , D Confocal microscopy analysis of plasma membrane active-β1 and active- α5β1 integrin expression and localization in living scr-shRNA- and sh-TFEB-ECs following incubation with anti-active β1 integrin (9EG7), active-α5β1 (SNAKA-51) and phalloidin-647 (scale bar: 25 µm). The bar graphs show the quantification of the mean intensity of active-β1 integrin and active- α5β1 ( n = 10 cells per condition pooled from three independent experiments; values as mean ± SEM; *** p < 0.0001 sh-TFEB-ECs versus scr-shRNA-ECs, as determined by Student’s t test)

Article Snippet: The following TaqMan assays were used: TFEB (Hs00292981_m1), ITGB1 s01127536_m1), ITGB3 (Hs01001469_m1), ITGA5 (Hs01547673_m1), ITGAV (Hs00233808_m1), CAV-1 (Hs00971716_m1), SCAP (Hs00378725_m1), SREBF-2 (Hs01081784_m1), HMGCR (Hs00168352_m1), and TBP (Hs00427620).

Techniques: Expressing, shRNA, Western Blot, Immunoprecipitation, Confocal Microscopy, Clinical Proteomics, Membrane, Incubation