Journal: Inflammatory bowel diseases

Article Title: miR-4728-3p functions as a tumor suppressor in ulcerative colitis-associated colorectal neoplasia through regulation of focal adhesion signaling

doi: 10.1097/MIB.0000000000001104

Figure Lengend Snippet: Expression correlation between miR-4728-3p and CAV1 (A), COL1A2 (B), and THBS2 (C) by microarray analysis. Quantitative real-time PCR was performed to validate expression of miR-4728-3p (D) as well as CAV1, COL1A2, and THBS2 (E) in the same 42 patients analyzed by microarray. Quantitative real-time PCR was also performed in 11 UC-associated cancers, nondysplastic adjacent tissue, and normal-appearing control tissues for miR-4728-3p (F) as well as CAV1, COL1A2, and THBS2 (G). Error bars represent standard error of the mean.

Article Snippet: Primary antibodies were diluted to 1:100 for CAV1 (Santa Cruz Biotechnology), 1:500 for COL1A2 (Novus Biologicals), 1:200 for THBS2 (Santa Cruz Biotechnology), and 1:5000 for beta actin (Sigma Aldrich).

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, Control

Journal: Inflammatory bowel diseases

Article Title: miR-4728-3p functions as a tumor suppressor in ulcerative colitis-associated colorectal neoplasia through regulation of focal adhesion signaling

doi: 10.1097/MIB.0000000000001104

Figure Lengend Snippet: A) Representative Western blots and B) quantitative expression by densitometry of CAV1, COL1A2, and THBS2 in the indicated colon cancer cell line following transfection with miR-4728-3p or scrambled oligo (n=3/treatment group). C) Firefly luciferase activity after transfection with miR-4728-3p and following treatment of indicated genes regulated by wild type or mutant 3′UTR (n=6/treatment group). To control for transfection efficiency, firefly luciferase activity was normalized to renilla luciferase activity. D) Representative images of wound healing in HCT116 cells treated with miR-4728-3p or a scrambled probe. E) Quantitative measurement of cell migration to assess wound closure in HCT116 cells treated with miR-4728-3p or a scrambled probe (values are means ± SD of 3 independent platings). F) Representative immunofluorescence images of vinculin and actin to assess focal adhesion complexes and actin cytoskeleton in HCT116 cells following treatment with miR-4728-3p or a scrambled probe. G) Focal adhesion intensity and H) area following treatment with miR-4728-3p or a scrambled probe (n=3 wells with 12 measurements each/treatment group). Error bars represent standard deviation.

Article Snippet: Primary antibodies were diluted to 1:100 for CAV1 (Santa Cruz Biotechnology), 1:500 for COL1A2 (Novus Biologicals), 1:200 for THBS2 (Santa Cruz Biotechnology), and 1:5000 for beta actin (Sigma Aldrich).

Techniques: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Mutagenesis, Control, Migration, Immunofluorescence, Standard Deviation

Journal: Bioactive Materials

Article Title: Bioinspired scaffold recapitulating chondrogenic ontogeny and microenvironment for functional cartilage regeneration

doi: 10.1016/j.bioactmat.2025.11.041

Figure Lengend Snippet: Biological functional analysis of tri-layer scaffold systems. A) Representative images of migrated cells stained with crystal violet at 48 h. B) Number of migrating cells in control and CE-SKP. C) Glycosaminoglycan concentrations in control and CE-SKP at 7 and 14 days. D) qPCR analysis for COL2, SOX9, and ACAN. E) Western blotting of COL2 and SOX9. F) Alizarin red staining of control and CPH. G) ALP activity of control and CPH. H) qPCR analysis for COL1, COL10, OCN, and RUNX2. I) Western blotting of COL10, COL1, and RUNX2. J) Representative images of migrated cells stained with crystal violet at 48 h. K) Number of migrating cells in control and P2G3. L) Scanning electron microscope micrographs of P2G3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: To evaluate the regeneration of cartilage, COL1 (1:100, HY- P81227 , MCE), COL2 (1:100, ARG20787 , Arigobio), and COL10 (1:100, BA2023, Boster) were used as a marker in immunohistochemistry (IHC) staining.

Techniques: Functional Assay, Staining, Control, Western Blot, Activity Assay, Microscopy

Journal: Bioactive Materials

Article Title: Bioinspired scaffold recapitulating chondrogenic ontogeny and microenvironment for functional cartilage regeneration

doi: 10.1016/j.bioactmat.2025.11.041

Figure Lengend Snippet: Regeneration of the femoral trochlea in a rabbit model. Representative images of IHC staining for A) COL1, B) COL10.

Article Snippet: To evaluate the regeneration of cartilage, COL1 (1:100, HY- P81227 , MCE), COL2 (1:100, ARG20787 , Arigobio), and COL10 (1:100, BA2023, Boster) were used as a marker in immunohistochemistry (IHC) staining.

Techniques: Immunohistochemistry

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Dioscin ameliorates peritoneal fibrosis by inhibiting epithelial-to-mesenchymal transition of human peritoneal mesothelial cells via the TLR4/MyD88/NF-κB signaling pathway

doi:

Figure Lengend Snippet: Dioscin inhibits LPS-induced fibrosis in HMrSV5 cells. Western blotting for detecting protein levels of α-SMA, collagen I and fibronectin. ###P < 0.001 compared with negative control group or dioscin (1.0 μg/ml) group. ***P < 0.001 compared with LPS group. NC, negative control; LPS, lipopolysaccharide; Dio, Dioscin; α-SMA, α-smooth muscle actin.

Article Snippet: Then the membranes were incubated with following primary antibodies: α-SMA (1:100, Proteintech Group), collagen I (1:100, Proteintech Group), fibronectin (1:250, Abcam), TLR4 (1:1000, Proteintech Group), MyD88 (1:1000, Proteintech Group), NF-κB (1:1000, Proteintech Group), TGF-β1 (1:1000, Proteintech Group), p-Smad2/Smad2 (1:1000, Proteintech Group), respectively.

Techniques: Western Blot, Negative Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Dioscin ameliorates peritoneal fibrosis by inhibiting epithelial-to-mesenchymal transition of human peritoneal mesothelial cells via the TLR4/MyD88/NF-κB signaling pathway

doi:

Figure Lengend Snippet: Dioscin inhibits EMT and fibrosis through TLR4/MyD88/NF-κB pathway in HMrSV5 cells. A. Western blotting for assessing protein levels of TLR4, MyD88, NF-κB, TGF-β1, p-Smad2, Smad2, α-SMA, collagen I and fibronectin. B. immunofluorescence assay for detecting expressions of α-SMA, collagen I and fibronectin in LPS+TLR4 group and LPS+TLR4+dioscin (0.5 μg/ml) group. ###P < 0.001 compared with negative control group or Ppicza group. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with LPS+ pPICZA group or LPS+TLR4 group. NC, negative control; LPS, lipopolysaccharide; Dio, Dioscin; α-SMA, α-smooth muscle actin; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor κB; TGF-β1, transforming growth factor-β1; TLR4, Toll-like receptor (TLR) 4.

Article Snippet: Then the membranes were incubated with following primary antibodies: α-SMA (1:100, Proteintech Group), collagen I (1:100, Proteintech Group), fibronectin (1:250, Abcam), TLR4 (1:1000, Proteintech Group), MyD88 (1:1000, Proteintech Group), NF-κB (1:1000, Proteintech Group), TGF-β1 (1:1000, Proteintech Group), p-Smad2/Smad2 (1:1000, Proteintech Group), respectively.

Techniques: Western Blot, Immunofluorescence, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Nano-hydroxyapatite/collagen film as a favorable substrate to maintain the phenotype and promote the growth of chondrocytes cultured in vitro

doi: 10.3892/ijmm.2018.3431

Figure Lengend Snippet: Immunohistochemical staining. Stronger positive expression (brown-yellow color) of Col II and weaker expression of Col I was observed in the nHC group compared with that in the HC and pure collagen groups, indicating that the chondrcocyte phenotype and biological functions could be maintained effectively when cells were cultured on nHC film for a long time period. HC, hydroxyapatite/collagen; nHC, nano-HC; calcein-AM/PI, acetoxymethyl/propidium iodide; TGF-β, transforming growth factor-β; COL-I, collagen type I; COL-II, collagen type II.

Article Snippet: Anti-COLIA1 and -COL1A2 antibodies and the immunohistochemistry (IHC) kit were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Expressing, Cell Culture

Journal: International Journal of Molecular Medicine

Article Title: Nano-hydroxyapatite/collagen film as a favorable substrate to maintain the phenotype and promote the growth of chondrocytes cultured in vitro

doi: 10.3892/ijmm.2018.3431

Figure Lengend Snippet: RT-qPCR analysis. Cartilage matrix relative gene expression was analyzed by RT-qPCR. The chondrocytes were seeded on the nHC, HC and pure collagen films and cultured for 6 days. Gene expression in cells relative to the control group was calculated by the 2 −ΔΔCq method using the β-actin gene as the internal control. The data are reported as the mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 vs. the collagen-based groups; # P<0.05, ## P<0.01 and ### P<0.001 for comparisons between the collagen-based groups and the TGF-β group. HC, hydroxyapatite/collagen; nHC, nano-HC; calcein-AM/PI, acetoxymethyl/propidium iodide; TGF-β, transforming growth factor-β; COL-I, collagen type I; COL-II, collagen type II; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Anti-COLIA1 and -COL1A2 antibodies and the immunohistochemistry (IHC) kit were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Quantitative RT-PCR, Gene Expression, Cell Culture, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Insulin receptor substrate‐1 time‐dependently regulates bone formation by controlling collagen Iα2 expression via miR‐342

doi: 10.1096/fj.201600445rr

Figure Lengend Snippet: Figure 3. Validation that murine miR-342 targets and controls the expression of Col1a2. A) Bioinformatics prediction of the binding region of mouse miR-342 (mmu-miR-342-3p) within the Col1a2 39-UTR. B) Sequence comparison of the Col1a2 39- UTR (WT) and the core sequence DM. These fragments were amplified, isolated, and inserted into the luciferase expression vector, pmirGLO, to examine luciferase reporter activity. C) In HEK293T cells transfected with WT Col1a2, an miR-342 mimic inhibited luciferase activity, and an miR-342 inhibitor enhanced luciferase activity. D) In HEK293 cells transfected with the DM, the miR-342 mimic lost the ability to inhibit luciferase activity. E) In osteogenic cells, the miR-342 inhibitor increased Col1a2 RNA (left) and protein (right) expression, but (F) an miR-342 mimic reduced Col1a2 protein (right) expression. *P , 0.05, compared to NC. Data are means 6 SD.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween [TBST; 50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and incubated overnight at 4°C in 3% nonfat dry milk in TBST supplemented with primary antibodies against COL1A2 (OAAF02899, 1:2000, Aviva Systems Biology, San Diego, CA, USA) and b-actin (bs-0061R, 1:1000, Bioss, Shanghai, China).

Techniques: Biomarker Discovery, Expressing, Binding Assay, Sequencing, Comparison, Isolation, Luciferase, Plasmid Preparation, Activity Assay, Transfection

Journal: The FASEB Journal

Article Title: Insulin receptor substrate‐1 time‐dependently regulates bone formation by controlling collagen Iα2 expression via miR‐342

doi: 10.1096/fj.201600445rr

Figure Lengend Snippet: Figure 4. Time-dependent expression of Col1a2 and miR-342 in Irs-1smla/smla mice and in bone marrow cells induced to osteogenic differentiation in vitro. A) Immunohistochemical analyses of bone tissue shows COL1A2 expression (brown stain) in BMSCs (triangles), and osteoblasts (arrows) from Irs-1+/+, Irs-1+/smla, and Irs-1smla/smla mice at 2 mo (left) and 12 mo (right) of age. At 2 mo of age, Irs-1smla/smla mice showed a higher COL1A2 expression in BMSCs than Irs-1+/+ mice. However, by 12 mo of age, COL1A2 expression levels were higher in osteoblasts from Irs-1smla/smla mice than in osteoblasts from Irs-1+/+ mice. B) Representative images of COL1A2 immunohistochemical stains with quantification of the osteoblast number on endosteal bone surface of the distal femora of 2- (2 M) and 12-mo-old (12 M) C57BL/6 mice. Es.N. COL1A2+/BS, number of COL1A2+ cells per endosteal bone surface (n = 5 per group). *P , 0.05, **P , 0.01, by 1-way ANOVA. C) Time course of Col1a2 mRNA and miR-342 expression during osteogenic induction in BMSCs isolated from WT mice. Col1a2 expression increased gradually, peaked after d 8, then decreased. Conversely, miR-342 expression increased up to d 2, reached a nadir after d 8, then increased. D) COL1A2 protein expression also increased gradually during osteogenic induction of BMSCs from WT mice, then, after d 8, protein levels decreased to d 16. E) During BMSCs osteogenic induction, ALP expression (black stain) increased, peaked on d 12, and then decreased. Expression of the osteocyte marker, DMP-1, was detected on d 12 and 16.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween [TBST; 50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and incubated overnight at 4°C in 3% nonfat dry milk in TBST supplemented with primary antibodies against COL1A2 (OAAF02899, 1:2000, Aviva Systems Biology, San Diego, CA, USA) and b-actin (bs-0061R, 1:1000, Bioss, Shanghai, China).

Techniques: Expressing, In Vitro, Immunohistochemical staining, Staining, Isolation, Marker

Journal: The FASEB Journal

Article Title: Insulin receptor substrate‐1 time‐dependently regulates bone formation by controlling collagen Iα2 expression via miR‐342

doi: 10.1096/fj.201600445rr

Figure Lengend Snippet: Figure 5. A Col1a2-specific siRNA and miR-342 mimic inhibited BMSC differentiation into osteoblasts and osteocyte-like cells. BMSCs from WT mice were transfected with either a Col1a2 siRNA/scrRNA or miR-342 mimic/inhibitor, then subjected to osteogenic differentiation. A) The Col1a2 siRNA, but not control (scrRNA), transfections inhibited the expression of COL1A2. B) (continued on next page)

Article Snippet: The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween [TBST; 50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and incubated overnight at 4°C in 3% nonfat dry milk in TBST supplemented with primary antibodies against COL1A2 (OAAF02899, 1:2000, Aviva Systems Biology, San Diego, CA, USA) and b-actin (bs-0061R, 1:1000, Bioss, Shanghai, China).

Techniques: Transfection, Control, Expressing

Journal: The FASEB Journal

Article Title: Insulin receptor substrate‐1 time‐dependently regulates bone formation by controlling collagen Iα2 expression via miR‐342

doi: 10.1096/fj.201600445rr

Figure Lengend Snippet: Figure 6. The relationship of COL1A2 expression in BMSCs, ALP staining in osteocytes, and in the capacity for in vitro osteogenesis in BMSCs derived from Irs-1smla/smla, Irs-1+/smla, and Irs-1+/+ mice. A) Western blots showing that compared to BMSCs from Irs-1+/smla and Irs-1+/+ mice, COL1A2 expression was clearly elevated in BMSCs from Irs-1smla/smla mice at 2 mo but not at 12 mo of age. B) Real-time PCR assays shows that the expression levels of miR-342 showed no significant statistical differences among Irs-1smla/smla, Irs-1+/smla, and Irs-1+/+ mice. C) ALP staining was also increased in osteocytes from Irs-1smla/smla mice compared to osteocytes from Irs-1+/smla and Irs-1+/+ mice. D) BMSCs from the Irs-1smla/smla mice showed low ALP staining after 4 d of osteogenesis induction and then differentiated into osteocyte-like cells after 12 d of osteogenesis induction. These osteocyte-like cells showed a more intense ALP staining than those from WT mice (see Fig. 4E). E) When BMSCs from the Irs-1smla/smla mice were transfected with a Col1a2 siRNA, they lost the ability to differentiate into osteocyte-like cells, even after 16 d of induction.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween [TBST; 50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and incubated overnight at 4°C in 3% nonfat dry milk in TBST supplemented with primary antibodies against COL1A2 (OAAF02899, 1:2000, Aviva Systems Biology, San Diego, CA, USA) and b-actin (bs-0061R, 1:1000, Bioss, Shanghai, China).

Techniques: Expressing, Staining, In Vitro, Derivative Assay, Western Blot, Real-time Polymerase Chain Reaction, Transfection