Journal: BioMed Research International

Article Title: Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

doi: 10.1155/2017/5316845

Figure Lengend Snippet: (a) Expression of three different isoforms of LH1, LH2, and LH3. The mRNA levels in samples of UF (F) and adjacent myometrial tissues (M) ( n = 20) were measured by quantitative real-time RT-PCR and were normalized to GAPDH mRNA levels. All values in fibroid tissues were described as fold differences compared to those measured in the corresponding myometrial tissues. Results are presented as mean ± SD. ∗ indicates a significant difference from control myometrium ( P < 0.05). (b) PCR amplification of different isoforms of LH2: cDNA from samples of UF and normal myometrial tissues were PCR amplified using primers for LH2a and LH2b. Aliquots of each amplified sample were subjected to electrophoresis in a 2% agarose gel containing ethidium bromide. The expected band sizes were 145 bp for LH2a and 208 bp for LH2b.

Article Snippet: Quantitative real-time RT-PCR was then carried out using Applied Biosystems 7500 real-time RT-PCR system (Applied Biosystems, Foster City, CA) using TaqMan gene expression assays (Applied Biosystems) for LH1 (assay ID Hs00609368_m1), LH2 (assay ID Hs00168688_m1), LH3 (assay ID Hs01126612_m1), LOX (assay ID Hs00184700_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (assay ID Hs99999905_m1).

Techniques: Expressing, Quantitative RT-PCR, Control, Amplification, Electrophoresis, Agarose Gel Electrophoresis

Journal: Biomacromolecules

Article Title: Harnessing the Biomimetic Effect of Macromolecular Crowding in the Cell-Derived Model of Clubfoot Fibrosis

doi: 10.1021/acs.biomac.4c00653

Figure Lengend Snippet: Relative mRNA expression of genes in clubfoot-derived fibroblasts (CF-M) after 24 h of cultivation without (Ctrl) or with (Fc18) a macromolecularly crowded environment. Genes of interest: COL1A1 , COL1A2 (collagen type I α-1 and α-2 chains); COL3A1 (collagen type III α-1 chain); COL6A3 (collagen type VI α-3 chain); PLOD1 , PLOD2 , PLOD3 (lysyl hydroxylase 1, 2, and 3); LOX (lysyl oxidase); P4THM (prolyl-4-hydroxylase); FN1 (fibronectin 1); TGFB1 (transforming growth factor-β 1); ACTA2 (α-smooth muscle actin); MMP2 , MMP9 (matrix metalloproteinases 2 and 9). Normalized against ACTB (β-actin) reference gene. Data presented as 2 ∧ (−ddCt), relative to Ctrl (calibrator). Mean + SD ( n = 3–5). Unpaired t test with Welch’s correction (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Real-time PCR was performed using 5xHOT FIREPol Probe qPCR Mix Plus (ROX) (08-14-00008, Solis BioDyne, Estonia) and TaqMan Gene Expression Assays (Life Technologies, USA) labeled with the FAM reporter dye specific to target the following human genes: the extracellular matrix proteins collagen type I α-1 chain ( COL1A1 ; Hs00164004_m1), collagen type I α-2 chain ( COL1A2 ; Hs00164099_m1), collagen type III α-1 chain ( COL3A1 ; Hs00943809_m1), collagen type VI α-3 chain ( COL6A3 ; Hs0091523_m1), and fibronectin 1 ( FN1 ; Hs01549976_m1), the enzymes catalyzing collagen post-translational modifications lysyl hydroxylase 1 ( PLOD1 ; Hs00386343_m1), lysyl hydroxylase 2 ( PLOD2 ; Hs01118184_m1), lysyl hydroxylase 3 ( PLOD3 ; Hs01126612_m1), lysyl oxidase ( LOX ; Hs00942483_m1), and prolyl 4-hydroxylase ( P4HTM ; Hs00977922_m1), the pro-fibrotic cytokine transforming growth factor β-1 ( TGFB1 ; Hs00998133_m1), the cytoskeletal protein α-smooth muscle actin ( ACTA2 ; Hs00909449_m1), the collagen degradation enzymes matrix metalloproteinase 2 and 9 ( MMP2, MMP9 ; Hs01548727_m1, Hs00957562_m1), and a reference gene β-actin ( ACTB ; Hs99999903_m1).

Techniques: Expressing, Derivative Assay

Journal: Biomacromolecules

Article Title: Harnessing the Biomimetic Effect of Macromolecular Crowding in the Cell-Derived Model of Clubfoot Fibrosis

doi: 10.1021/acs.biomac.4c00653

Figure Lengend Snippet: Comparison of contracted (M-side) and noncontracted (L-side) of clubfoot tissue—complementary data. (A) Demonstrative images of immunohistochemical (IHC) staining and signal detection. Staining of collagen type III and VI, lysyl hydroxylases 1, 2, and 3 (LH1, LH2, LH3). The red areas in the ST (signal threshold) columns represent the positive pixels after thresholding. An image analyzer was then used to measure the percentage of the area with a positive signal from the total area. E+ Extracellular positivity, N+ Nuclear positivity, C+ Cytosolic positivity , N/A Not Assessed. Scale bar = 20 μm. (B) The percentage of COL3, COL6, LH2, and LH3 positive area after IHC signal quantification. LH1 was not detectable. Scatter plots, median with 95% CI ( n = 10). Paired t test (** p < 0.01, *** p < 0.001).

Article Snippet: Real-time PCR was performed using 5xHOT FIREPol Probe qPCR Mix Plus (ROX) (08-14-00008, Solis BioDyne, Estonia) and TaqMan Gene Expression Assays (Life Technologies, USA) labeled with the FAM reporter dye specific to target the following human genes: the extracellular matrix proteins collagen type I α-1 chain ( COL1A1 ; Hs00164004_m1), collagen type I α-2 chain ( COL1A2 ; Hs00164099_m1), collagen type III α-1 chain ( COL3A1 ; Hs00943809_m1), collagen type VI α-3 chain ( COL6A3 ; Hs0091523_m1), and fibronectin 1 ( FN1 ; Hs01549976_m1), the enzymes catalyzing collagen post-translational modifications lysyl hydroxylase 1 ( PLOD1 ; Hs00386343_m1), lysyl hydroxylase 2 ( PLOD2 ; Hs01118184_m1), lysyl hydroxylase 3 ( PLOD3 ; Hs01126612_m1), lysyl oxidase ( LOX ; Hs00942483_m1), and prolyl 4-hydroxylase ( P4HTM ; Hs00977922_m1), the pro-fibrotic cytokine transforming growth factor β-1 ( TGFB1 ; Hs00998133_m1), the cytoskeletal protein α-smooth muscle actin ( ACTA2 ; Hs00909449_m1), the collagen degradation enzymes matrix metalloproteinase 2 and 9 ( MMP2, MMP9 ; Hs01548727_m1, Hs00957562_m1), and a reference gene β-actin ( ACTB ; Hs99999903_m1).

Techniques: Comparison, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Biomacromolecules

Article Title: Harnessing the Biomimetic Effect of Macromolecular Crowding in the Cell-Derived Model of Clubfoot Fibrosis

doi: 10.1021/acs.biomac.4c00653

Figure Lengend Snippet: Relative mRNA expression of genes for collagen and collagen-cross-linking enzymes in clubfoot-derived fibroblasts (CF-M) after 24 h of cultivation in noncrowded Ctrl or macromolecularly crowded Fc18 media with or without the addition of minoxidil (MXD). Genes of interest: PLOD1 , PLOD2 , PLOD3 (lysyl hydroxylase 1, 2, and 3); COL1A1 , COL1A2 (collagen type I α-1 and α-2 chains). Normalized against ACTB (β-actin) reference gene. Data presented as 2 ∧ (−ddCt), relative to Ctrl (calibrator). Mean + SD ( n = 4). Two-Way ANOVA, Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Real-time PCR was performed using 5xHOT FIREPol Probe qPCR Mix Plus (ROX) (08-14-00008, Solis BioDyne, Estonia) and TaqMan Gene Expression Assays (Life Technologies, USA) labeled with the FAM reporter dye specific to target the following human genes: the extracellular matrix proteins collagen type I α-1 chain ( COL1A1 ; Hs00164004_m1), collagen type I α-2 chain ( COL1A2 ; Hs00164099_m1), collagen type III α-1 chain ( COL3A1 ; Hs00943809_m1), collagen type VI α-3 chain ( COL6A3 ; Hs0091523_m1), and fibronectin 1 ( FN1 ; Hs01549976_m1), the enzymes catalyzing collagen post-translational modifications lysyl hydroxylase 1 ( PLOD1 ; Hs00386343_m1), lysyl hydroxylase 2 ( PLOD2 ; Hs01118184_m1), lysyl hydroxylase 3 ( PLOD3 ; Hs01126612_m1), lysyl oxidase ( LOX ; Hs00942483_m1), and prolyl 4-hydroxylase ( P4HTM ; Hs00977922_m1), the pro-fibrotic cytokine transforming growth factor β-1 ( TGFB1 ; Hs00998133_m1), the cytoskeletal protein α-smooth muscle actin ( ACTA2 ; Hs00909449_m1), the collagen degradation enzymes matrix metalloproteinase 2 and 9 ( MMP2, MMP9 ; Hs01548727_m1, Hs00957562_m1), and a reference gene β-actin ( ACTB ; Hs99999903_m1).

Techniques: Expressing, Derivative Assay