Journal: Cardiovascular Research

Article Title: Regulation of Ptbp1-controlled alternative splicing of pyruvate kinase muscle by liver kinase B1 governs vascular smooth muscle cell plasticity in vivo

doi: 10.1093/cvr/cvae187

Figure Lengend Snippet: Histological analysis of the aortas from WT and Lkb1 SMiKO mice. ( A ) H&E, elastin, and Masson trichrome stains of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.0–1.5 months post-TAM induction. Scale bars: 200 µm. ( B ) H&E staining and immunofluorescence staining (α-actin) of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.0–1.5 months post-TAM induction. Scale bars: 20 and 50 µm, respectively. ( C ) Quantification of the medial wall thickness of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.5 months post-TAM induction. Data represent mean ± SEM ( n = 6 for WT mice and n = 8 for Lkb1 SMiKO mice). ( D ) Quantification of the internal and external elastic laminae (IEL and EEL, respectively) perimeters of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.5 and 4.5 months post-TAM induction. Data represent mean ± SEM ( n = 6 for WT mice and n = 8 for Lkb1 SMiKO mice). ( E ) H&E, elastin, and Masson stains of the thoracic aortas from WT and Lkb1 SMiKO mice at 4.5 months post-TAM induction. Scale bars: 200 µm. Boxes indicate fields shown at higher magnification in images to the immediate right. ( F ) Alcian Blue, Safranin O, and Von Kossa staining of the thoracic aortas from WT and Lkb1 SMiKO mice at 4.5 months post-TAM induction. Scale bars: 200 µm. Boxes indicate fields shown at higher magnification in images to the immediate right. ( G and H ) Immunofluorescence staining for α-actin ( G ), SM22α ( G ), and Col2a1 ( H ) of the thoracic aortas from WT and Lkb1 SMiKO mice at 4.5 months post-TAM induction ( n = 6 mice per genotype). Where indicated, nuclei were counterstained with DAPI. Dashed lines denote the IEL and EEL of the aortas. Scale bars: 200 µm. Boxes indicate fields shown at higher magnification in images to the immediate right. P -values were determined using Student’s t -test ( C and D ). For all panels, * P < 0.05; ** P < 0.01.

Article Snippet: Briefly, OCT-embedded sections were washed with phosphate-buffered saline (PBS), fixed with acetone at 4°C for 15 min, and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with goat serum (protein block) for 30 min, the sections were incubated with primary antibodies against α-actin (Abcam, Cat #ab7817, 1:200), SM22α (Abcam, Cat #ab14106, 1:200), Col2A1 (Boster, Cat #PA2141-1, 1:200), and Sox9 (Millipore, Cat #ABE2868, 1:500) overnight.

Techniques: Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells

doi: 10.1038/s41598-020-67836-3

Figure Lengend Snippet: List of primers used in RT-qPCR for chondrogenic differentiation genes.

Article Snippet: The supernatant of media was measured using ELISA kits for aggrecan (LS-F5714, LSBio, Seattle, WA, USA), COL2A1 (type II collagen, LS-F26742, LSBio), type I collagen (LS-F26726, LSBio) and MMP13 (LS-F26160, LSBio) according to protocols provided by the manufacturer.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells

doi: 10.1038/s41598-020-67836-3

Figure Lengend Snippet: Gene expression analysis of hMSC beads. ( A ) mRNA expression levels of (a) Sox9 , (b) type II collagen , (c) aggrecan and (d) type I collagen in control I group (mixture of fibrin, hMSCs, and thrombin cultured in basal medium), control II group (mixture of fibrin, hMSCs, and thrombin cultured in chondrogenic differentiation medium), and the atelocollagen group (mixture of fibrin, atelocollagen, hMSCs, and thrombin cultured in chondrogenic differentiation medium) at 7, 14 and 21 days after in vitro culture. (a) The expression level of Sox9 in the atelocollagen group was highest 18.1-fold on day 7 to 23.9-fold on day 21 of culture and remained high during the culture period. On day 21, Sox9 expression was significantly higher compared to day 7 (***, orange indication). The expression of Sox9 results increased early on day 7 compared to the other chondrogenic differentiation markers. (b) Type II collagen expression was highly upregulated 254.3-fold on day 14 and 532.6-fold on day 21 of culture in the atelocollagen group. Among the other chondrogenic differentiation markers, the type II collagen expression value was the highest on day 21 in the atelocollagen group. Expression values on day 21 were significant compared to day 7 (***, orange indication). (c) The aggrecan expression of the atelocollagen group increased rapidly 0.3-fold on day 7 to 176.9-fold on day 21, and a significant value was shown (***, orange indication). (d) Nevertheless, type I collagen expression was increased 3.7- to 3.9-fold in the control II group and 2.9- to 3.5-fold in the atelocollagen group on days 7 and 14. However, on day 21, type I collagen expression decreased 1.5-fold in the control II group and 1.3-fold in the atelocollagen group. Decreased values on day 21 were significant when compared with day 7 (***, orange indication). * Black indication: Compare expression values of groups on the same day. * Orange indication: Comparison of expression values by day 7 (early stage). ( B ) To evaluate the mRNA expression levels of (a) Type X Collagen (COL10A1) , (b) Runx2, (c) ALP, and (d) Osterix in the control I, control II and atelocollagen groups on day 21 after in vitro culture. (a) CoL10A1 expression was decreased 0.58-fold on day 21, which was significant in the atelocollagen group compared to the control I group (*). (b) Runx2 expression was slightly increased by 1.19-fold in the control II group. The atelocollagen group expression was significantly decreased by 0.57-fold in the atelocollagen group compared to the control I group (***). Expression in the control II group was significantly increased by 1.52-fold in (d) Osterix (***) and 1.49-fold in (c) ALP marker (*) compared to the control I group. Expression in the atelocollagen group was decreased by 0.56-fold in Osterix (**) and 1.35-fold in ALP marker (*). n = 3 in each group. # P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The supernatant of media was measured using ELISA kits for aggrecan (LS-F5714, LSBio, Seattle, WA, USA), COL2A1 (type II collagen, LS-F26742, LSBio), type I collagen (LS-F26726, LSBio) and MMP13 (LS-F26160, LSBio) according to protocols provided by the manufacturer.

Techniques: Gene Expression, Expressing, Control, Cell Culture, In Vitro, Comparison, Marker

Journal: Scientific Reports

Article Title: Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells

doi: 10.1038/s41598-020-67836-3

Figure Lengend Snippet: Expression of cartilage matrix-related protein in hMSC beads. Protein expression levels in the culture supernatants from control I group (mixture of fibrin, hMSCs, and thrombin cultured in basal medium), control II group (mixture of fibrin, hMSCs, and thrombin cultured in chondrogenic differentiation medium), and atelocollagen group (mixture of fibrin, atelocollagen, hMSCs, and thrombin cultured in chondrogenic differentiation medium) were analyzed by ELISA. ( A ) Aggrecan levels in the culture media of the atelocollagen group showed higher expression as compared to both the control I and II groups (***). ( B ) COL2A1 expression showed similar trends to aggrecan expression (***). ( C ) Type I collagen expression was slightly increased in the control II group (*), however, the expression of the atelocollagen group was not ( # ). ( D ) MMP13 was only slightly expressed in the atelocollagen group. The results were significant compared to the control I group (*). n = 3 in each group. # P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The supernatant of media was measured using ELISA kits for aggrecan (LS-F5714, LSBio, Seattle, WA, USA), COL2A1 (type II collagen, LS-F26742, LSBio), type I collagen (LS-F26726, LSBio) and MMP13 (LS-F26160, LSBio) according to protocols provided by the manufacturer.

Techniques: Expressing, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells

doi: 10.1038/s41598-020-67836-3

Figure Lengend Snippet: Histological analysis of chondrogenic markers in hMSC beads. ( A ) Staining of fibrin, thrombin, and atelocollagen in the absence of cells. The gel beads without cells were cultured in chondrogenic differentiation medium on day 21. (a) Hematoxylin–eosin demonstrated weak eosin staining (weak pink color) and one large mass without empty space. (b) Weak Alcian blue pH 2.5 and (c) toluidine blue staining was observed. (e) Type I and (d) type II collagen staining did not demonstrate brown expression. Scale bar: 20 µm. ( B ) Staining of fibrin, thrombin, and atelocollagen with cells. Representative histological images of the control I group (mixture of fibrin, hMSCs, and thrombin cultured in basal medium), control II group (mixture of fibrin, hMSCs, and thrombin cultured in chondrogenic differentiation medium), and atelocollagen group (mixture of fibrin, atelocollagen, hMSCs, and thrombin cultured in chondrogenic differentiation medium) at 21 days after culture. Hematoxylin–eosin staining showed well mixed cells and a structure similar to lacunae (k, black arrow) compared to the other groups (a and f). Alcian blue pH 2.5 and toluidine blue staining showed expression around cells in the atelocollagen group (l and m), and the control I and II groups contained weak expression in the densely packed cells (b and g, c and h). Type II collagen was highly expressed around chondrocyte-like cells in the atelocollagen group (n) and weakly expressed in the control II group (i) compared to the control I group (d). Type I collagen staining was not observed in the control II and atelocollagen groups (j and o). Nevertheless, gel beads of the control I group were weakly expressed (e). Scale bar; 20 µm.

Article Snippet: The supernatant of media was measured using ELISA kits for aggrecan (LS-F5714, LSBio, Seattle, WA, USA), COL2A1 (type II collagen, LS-F26742, LSBio), type I collagen (LS-F26726, LSBio) and MMP13 (LS-F26160, LSBio) according to protocols provided by the manufacturer.

Techniques: Staining, Cell Culture, Expressing, Control

Journal: Bioengineering

Article Title: Comparison of Bioengineered Scaffolds for the Induction of Osteochondrogenic Differentiation of Human Adipose-Derived Stem Cells

doi: 10.3390/bioengineering11090920

Figure Lengend Snippet: hASC survival and differentiation in GelMA hydrogels. ( A ) CCK8 assay of hASCs encapsulated in the GelMA hydrogel at different time points (1 week intervals). Results are reported as mean ± SEM of n = 3 samples/group. ( B ) Representative confocal imaging of Dead/live fluorescence staining of hASCs in the GelMA hydrogel after 3 weeks of culture. Red cells are dead while green cells are alive. ( C ) Alcian blue staining of GelMA hydrogel after 24 h and 3 weeks of culture. ( D ) Alizarin red staining of GelMA hydrogel after 24 h and 3 weeks of culture. ( E , F ) Confocal imaging of immunofluorescence for COL2A1 (green) and OCN (red) in hASCs encapsulated in GelMA hydrogel after 3 weeks of culture.

Article Snippet: Briefly, samples were incubated with rabbit polyclonal anti-collagen type II alpha 1 (COL2A1) antibody (Merck) and rabbit polyclonal anti-osteocalcin (OCN) antibody (Thermo Fisher Scientific) for 1 h at room temperature.

Techniques: CCK-8 Assay, Imaging, Fluorescence, Staining, Immunofluorescence

Journal: Bioengineering

Article Title: Comparison of Bioengineered Scaffolds for the Induction of Osteochondrogenic Differentiation of Human Adipose-Derived Stem Cells

doi: 10.3390/bioengineering11090920

Figure Lengend Snippet: hASC survival and differentiation in PEGDA scaffold. ( A ) CCK8 assay of hASCs seeded in the PEGDA scaffold at different time points (1 week intervals). Results are reported as mean ± SEM of n = 3 samples/group. T test: * p < 0.05. ( B ) Representative confocal imaging of Dead/live fluorescence staining of hASCs seeded in the PEGDA scaffold after 3 weeks of culture. ( C ) Confocal imaging of immunofluorescence for COL2A1 (green) and OCN (red) in hASCs seeded in the PEGDA scaffold after 3 weeks of culture.

Article Snippet: Briefly, samples were incubated with rabbit polyclonal anti-collagen type II alpha 1 (COL2A1) antibody (Merck) and rabbit polyclonal anti-osteocalcin (OCN) antibody (Thermo Fisher Scientific) for 1 h at room temperature.

Techniques: CCK-8 Assay, Imaging, Fluorescence, Staining, Immunofluorescence

Journal: Bioengineering

Article Title: Comparison of Bioengineered Scaffolds for the Induction of Osteochondrogenic Differentiation of Human Adipose-Derived Stem Cells

doi: 10.3390/bioengineering11090920

Figure Lengend Snippet: hASC survival and differentiation in celery-based scaffold. ( A ) SEM imaging of a single hASC inside a niche of the celery-based scaffold. ( B ) CCK8 assay of hASCs seeded in the celery-based scaffold at different time points (1 week intervals). Results are reported as mean ± SEM of n = 3 samples/group. T test: * p < 0.05. ( C ) Representative confocal imaging of Dead/live fluorescence staining of hASCs seeded in the scaffold after 3 weeks of culture. ( D ) Confocal 3D stack and ( E ) the projection of hASC distribution inside the scaffold. ( F ) Confocal imaging of immunofluorescence for COL2A1 (green) and OCN (red) in hASCs seeded in the PEGDA scaffold after 3 weeks of culture.

Article Snippet: Briefly, samples were incubated with rabbit polyclonal anti-collagen type II alpha 1 (COL2A1) antibody (Merck) and rabbit polyclonal anti-osteocalcin (OCN) antibody (Thermo Fisher Scientific) for 1 h at room temperature.

Techniques: Imaging, CCK-8 Assay, Fluorescence, Staining, Immunofluorescence

Journal: Biomolecules

Article Title: COL2A1 and Caspase-3 as Promising Biomarkers for Osteoarthritis Prognosis in an Equus asinus Model

doi: 10.3390/biom10030354

Figure Lengend Snippet: ( A ) Western blot membrane representing the specific band for COL2A1 protein from the synovial fluid; located at MWT, about 65 KDa. (M): pre-stained protein marker; lanes 1, 2 representing samples at day 0 (control); lanes 3, 4 representing samples for the 2nd month; lane 5: samples at the 3rd month; lanes 6 and 7 samples at the 5th month; and lanes 8 and 9 corresponding to the 7th month. ( B ) Graphical representation for the level of COL2A1 protein expression level relative to total protein Coomassie blue staining) as quantified by densitometric analysis of the bands. Data presented as the mean ± SEM for triplicate samples ( n = 3). Values with the same letters are non-significantly different ( p > 0.05) while the values of different letters are significantly different ( p < 0.05).

Article Snippet: The primary antibody used was polyclonal anti-type II collagen, COL2A1 antibody (Chongqing Biospes, cat# YPA1669), and HRP-conjugate goat anti-rabbit IgG as a secondary antibody (Chongqing Biospes, cat#BSA1013).

Techniques: Western Blot, Membrane, Staining, Marker, Control, Expressing