normal human fetal lung fibroblasts  (ATCC)

Journal: Pharmaceutics

Article Title: Targeting Lysyl Oxidase-like 2: A Therapeutic Strategy for Idiopathic Pulmonary Fibrosis with a Novel Indolizine Derivative

doi: 10.3390/pharmaceutics18050554

Figure Lengend Snippet: Compound #765 inhibits LOXL2 activity and TGF-β1-induced collagen remodeling in lung fibroblasts. ( A , B ) Effects of Compound #765 in the absence of TGF-β1. ( A ) LOXL2 enzymatic activity in MRC5 fibroblast supernatants after 72 h of treatment with Compound #765 or BAPN, showing a dose-dependent decrease. ( B ) Soluble collagen levels were significantly reduced following Compound #765 treatment. ( C – F ) Effects of Compound #765 under TGF-β1 stimulation (2.5 ng/mL). ( C ) Representative images of collagen gel contraction in MRC5 fibroblasts with TGF-β1 in the presence or absence of Compound #765 (100 nM or 200 nM). Gel contraction assays were performed in 24-well plates. The gels were then carefully detached, transferred to 60 mm dishes, and images were acquired using a Fusion Solo 6S system equipped with an eVo-6 camera. ( D ) Quantification of gel contraction at 0, 24, 48 h using ImageJ. ( E ) Immunoblot analysis of fibrosis-related proteins (α-SMA, LOXL2, and fibronectin (FN)) in TGF-β1-treated MRC5 cells with or without Compound #765 . ( F ) Densitometric quantification of protein expression normalized to GAPDH. Overall, Compound #765 inhibited LOXL2 enzymatic activity, reduced collagen secretion, suppressed TGF-β1-induced collagen contraction, and downregulated α-SMA and LOXL2 while FN levels remained largely unchanged. Data are presented as mean ± SD ( n = 3). LOXL2, lysyl oxidase-like 2; TGF-β1, transforming growth factor-beta 1; MRC5, human lung fibroblast cell line; α-SMA, alpha-smooth muscle actin; FN, fibronectin.

Article Snippet: Human fetal lung fibroblasts (MRC5; ATCC, Manassas, VA, USA) were cultured in Minimum Essential Medium (MEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin/streptomycin (P/S; Hyclone).

Techniques: Activity Assay, Western Blot, Expressing

Journal: Pharmaceutics

Article Title: Targeting Lysyl Oxidase-like 2: A Therapeutic Strategy for Idiopathic Pulmonary Fibrosis with a Novel Indolizine Derivative

doi: 10.3390/pharmaceutics18050554

Figure Lengend Snippet: Compound #765 inhibits fibrosis-related protein expression and collagen contraction in human lung fibroblasts. Normal lung fibroblasts (LL24) and IPF-derived fibroblasts (LL97A) were stimulated with TGF-β1 (5 ng/mL) and treated with Compound #765 . ( A ) Representative Western blot showing α-SMA expression after treatment with Compound #765 (100 nM) for 24, 48, and 72 h. GAPDH served as a loading control. ( B ) Collagen gel contraction assay in LL24 and LL97A fibroblasts treated with Compound #765 (100, 250, and 500 nM) following TGF-β1 stimulation. Contraction was quantified at the indicated time points. Data are presented as mean ± SD ( n = 3). ( C ) Representative immunofluorescence images of collagen I (Col-I), α-SMA, and FN after 72 h of treatment with TGF-β1 (5 ng/mL) and Compound #765 (100, 250, and 500 nM). Nuclei were counterstained with Hoechst 33342 (blue). Images were acquired at 40× magnification. α-SMA, alpha-smooth muscle actin; LL24, a normal lung fibroblast cell line; IPF, idiopathic pulmonary fibrosis; LL97A, an IPF-derived fibroblast cell line; TGF-β1, transforming growth factor-beta 1; Col-I, collagen I; FN, fibronectin.

Article Snippet: Human fetal lung fibroblasts (MRC5; ATCC, Manassas, VA, USA) were cultured in Minimum Essential Medium (MEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin/streptomycin (P/S; Hyclone).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Collagen Gel Contraction Assay, Immunofluorescence

Journal: Pharmaceutics

Article Title: Targeting Lysyl Oxidase-like 2: A Therapeutic Strategy for Idiopathic Pulmonary Fibrosis with a Novel Indolizine Derivative

doi: 10.3390/pharmaceutics18050554

Figure Lengend Snippet: Compound #765 suppresses TGF-β1-induced fibrotic gene expression in human lung fibroblasts. Normal lung fibroblasts (LL24) and IPF-derived fibroblasts (LL97A) were stimulated with TGF-β1 (5 ng/mL) and treated with Compound #765 (100, 250, and 500 nM) for 24, 48, and 72 h. mRNA expression of fibrosis-associated genes was measured by quantitative real-time PCR. ( A ) Relative mRNA levels of ACTA2, COL1A1, and LOXL2 in LL24 cells. ( B ) Relative mRNA levels of ACTA2, COL1A1, and LOXL2 in LL97A cells. Compound #765 significantly reduced TGF-β1-induced gene expression in dose- and time-dependent manner in both cells. Data are presented as mean ± SD ( n = 3–5). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001, vs. TGF-β1 + DMSO group. ns, not significant. ACTA2, actin alpha 2, smooth muscle; COL1A1, collagen type I alpha 1.

Article Snippet: Human fetal lung fibroblasts (MRC5; ATCC, Manassas, VA, USA) were cultured in Minimum Essential Medium (MEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin/streptomycin (P/S; Hyclone).

Techniques: Gene Expression, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction