control fibroblasts  (ATCC)

Journal: Journal of Extracellular Vesicles

Article Title: Cholesterol Deficiency Directs Autophagy‐Dependent Secretion of Extracellular Vesicles

doi: 10.1002/jev2.70218

Figure Lengend Snippet: Cholesterol depletion promotes small extracellular vesicle release. (A) Cholesterol metabolic reactions targeted with small molecule inhibitors (red text). Rare diseases of cholesterol biosynthesis (blue text) arise from genetic mutations to indicated enzymes (grey). (B) GC‐MS analysis of human fibroblasts shows Smith–Lemli–Opitz syndrome (SLOS) and lathosterolosis (LATH) are cholesterol‐deficient and accumulate sterol precursors (mean ± SEM; n = 3 biological replicates from 3 independent experiments). (C) Small extracellular vesicle (sEV) release is elevated in SLOS and LATH fibroblasts relative to controls (mean ± SEM; n = 4 biological replicates from 4 independent experiments). One‐way ANOVA (F2,9 = 10.22, p ≤ 0.0048), Dunnett's multiple comparisons (* p ≤ 0.05; ** p ≤ 0.005). (D) Nanoparticle tracking analysis (NTA) of sEV size from HEK293T, HaCaT and HMC3 cells (mean; n = 4 biological replicates from 4 independent experiments). (E) Representative transmission electron microscopy (TEM) image of sEVs isolated from HEK293T cells. Scale bar, 100 nm. (F) CD9 immunoblot in HEK293T‐isolated sEVs following treatment with respective cholesterol biosynthesis inhibition. (G) Inhibition of cholesterol biosynthesis in HEK293T cells results in increased sEV release (mean ± SEM; n = 4 biological replicates from 4 independent experiments). One‐way ANOVA (F5,18 = 20.51, p < 0.0001), Dunnett's multiple comparisons test (** p ≤ 0.005, **** p < 0.0001). (H) Regression analysis of sEVs from HEK293T cells reveals a strong negative correlation between sEV secretion and cellular cholesterol levels. Linear regression (F1,4 = 15.17, R 2 = 0.7913, p ≤ 0.0176).

Article Snippet: Control fibroblasts were purchased commercially (ATCC, CRL‐2522).

Techniques: Gas Chromatography-Mass Spectrometry, Transmission Assay, Electron Microscopy, Isolation, Western Blot, Inhibition

Journal: Medicina

Article Title: Evaluation of TAM Receptor Targeting in Pathophysiology of Idiopathic Pulmonary Fibrosis

doi: 10.3390/medicina61101837

Figure Lengend Snippet: Relative mRNA expression levels of Axl, Tyro3, and Gas6 genes in IPF FBs and HPFs quantified by RT-qPCR. No Mer expression was detected in both fibroblast types. TBP gene was used as housekeeping gene. N = 4.

Article Snippet: Control human pulmonary fibroblasts (HPFs, C12360 , PromoCell, St. Louis, MO, USA) were cultured in 25 mM glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin solution, and cells were used between passages 7 and 15.

Techniques: Expressing, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Bioenergetic Signatures of DLD Deficiency: Dissecting PDHc- and α-KGDHc-Linked Defects

doi: 10.3390/antiox15010019

Figure Lengend Snippet: Mitochondrial respiration is impaired in fibroblasts derived from patients with DLD deficiency. Mitochondrial oxygen consumption was assessed in controls (Ctrl1 and Ctrl2) and patient (Pt1–Pt6) fibroblasts using high-resolution respirometry (Oroboros O2k). ( A ) Routine respiration; ( B ) maximal respiration calculated as the difference between FCCP-stimulated and α-chaconine–permeabilized rates; ( C ) complex I-linked respiration (NADH-linked respiration, N-pathway), calculated as the difference between ADP and α-chaconine; ( D ) complex II-linked respiration (NS-pathway) calculated as the difference between respiration after rotenone and α-chaconine addition; ( E ) effect of complex I inhibition, calculated as the difference between FCCP-stimulated and rotenone-inhibited respiration; and ( F ) complex I/complex II respiration ratio (complex I-linked activity divided by complex II-linked activity). Each open circle represents an independent experimental run (N = 4–8 repeats per sample). All data were normalized to cell number. Statistical analysis was performed using the Mann–Whitney U test. * p < 0.05 vs. Ctrl1; numerical p values (0.05 < p < 0.1) are indicated on the plots. Abbreviations: Ctrl, control; Pt, patient.

Article Snippet: Dermal fibroblast primary cell lines from six genetically confirmed patients with DLD deficiency were obtained from the Pediatric Metabolic Disease Unit, Sheba Medical Center (IRB# SMC-21-8644, Figure 1, Table 1, and ), as well as two control cell lines: a control human dermal fibroblast cell line was purchased from ATCC (PCS-201-012; Ctrl 1, Manassas, VA, USA), and a primary cell line from a 39-year-old healthy male (Ctrl 2).

Techniques: Derivative Assay, Inhibition, Activity Assay, MANN-WHITNEY, Control