Journal: NPJ Science of Food

Article Title: Haematococcus pluvialis ameliorates renal fibrosis by restoring mitophagy via PINK1-Parkin-p62-LC3 signaling

doi: 10.1038/s41538-025-00654-x

Figure Lengend Snippet: A , B Histopathological evaluation of renal cortical tissues through HE, Masson, and PAS staining (scale bar: 20 μm). Tubular regions (A, 63×) and glomerular structures (B, 40×). C Quantitative analysis of collagen fiber deposition (blue) in tubular regions via Masson staining using ImageJ ( n = 8). D Glomerular collagen content quantified by Masson staining and glomerular MMI measured by PAS staining ( n = 8), with magenta indicating glycogen deposition. E Blue: nuclei; Brown: FN1 and COL4A. Positive expression of FN1 and COL4A proteins in renal cortical tissues was detected by IHC staining (magnification: 63×, scale bar: 20 μm). F Quantitative analysis of positive areas (brown) in IHC results using ImageJ ( n = 8). G Western blot analysis and gray value quantification of α-SMA, FN1, and COL4A expression in renal cortical tissues from different experimental groups. β-Actin and β-Tubulin was used as a loading control. Gray value quantification of Western blotting results using ImageJ ( n = 3). H Functional enrichment analysis of FN1 gene and COL4A2 gene via RNA-seq profiling. * p < 0.05 vs. Sham group, # p < 0.05 vs. UUO group.

Article Snippet: LC3 Polyclonal antibody (14600-1-AP), p62 Polyclonal antibody (80294-1-RR), PINK1 Polyclonal antibody (23274-1-AP), Parkin Polyclonal antibody (14060-1-AP), E-cadherin Polyclonal antibody (20874-1-AP), N-cadherin Polyclonal antibody (No.: 22018-1-AP), Vimentin Polyclonal antibody (10366-1-AP), COL4A2 Polyclonal antibody (55131-1-AP), FN1 Monoclonal antibody (66042-1-Ig), β-Actin Polyclonal antibody (20536-1-AP), and α-Tubulin Polyclonal antibody (11224-AP) were from Proteintech group Co., Ltd (Wuhan, China).

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Control, Functional Assay, RNA Sequencing

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Primers used for quantitative PCR analyses.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Narrowing down of the domain involved in the induction of COL1A1 expression in LX-2 cells. (A) Overexpression of both full-length fibrinogen domain of TNX [TNX-FG] and ITGA11 failed to induce the expression of fibrosis marker genes, including ACTA2, COL1A1 and TGFB1 . LX-2 cells were transfected with expression vectors for hTNX-FG (lane 2), ITGA11 (lane 3) and hTNX-FG and ITGA11 (lane 4) in DMEM/0.5% FBS. (B) Induction of COL1A1 expression by overexpression of both hTNX-FGF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGF (lane 2), hTNX-FGL (lane 3), ITGA11 (lane 4), hTNX-FGF and ITGA11 (lane 5) and hTNX-FGL and ITGA11 (lane 6) in DMEM/0.5% FBS. (C) Induction of COL1A1 expression by overexpression of both hTNX-FGFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFF (lane 2), hTNX-FGFL (lane 3), ITGA11 (lane 4), hTNX-FGFF and ITGA11 (lane 5) and hTNX-FGFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (D) Induction of COL1A1 expression by overexpression of both hTNX-FGFFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFFF (lane 2), hTNX-FGFFL (lane 3), ITGA11 (lane 4), hTNX-FGFFF and ITGA11 (lane 5) and hTNX-FGFFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (A-D) As a control, RNA from the cells without transfection (lane 1) was used. The cell lysate was prepared 48 h after transfection and then RNA was purified. Subsequently, the expression levels of ACTA2, COL1A1 and TGFB1 were examined by reverse transcription-quantitative PCR. The expression level of each gene in the control was set to 1.0, and the relative expression level of each gene compared with that of the control is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1), one-way ANOVA with Dunnett's post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; hTNX-FG, fibrinogen-related domain of human tenascin-X; hTNX-FGF, first half of hTNX-FG; hTNX-FGL, latter half of hTNX-FG; hTNX-FGFF, first half of hTNX-FGF; hTNX-FGFL, latter half of hTNX-FGF; hTNX-FGFFF, first half of hTNX-FGFF; hTNX-FGFFL, latter half of hTNX-FGFF.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Expressing, Over Expression, Marker, Transfection, Control, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Identification of the minimal sequence responsible for induction of COL1A1 expression in LX-2 cells. (A) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGFFFL (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGFFFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (B) Overexpression of both hTNX-FGpeptide2-5 and ITGA11 did not cause induction of COL1A1 expression. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGpeptide2-5 (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGpeptide2-5 and ITGA11 (lane 6) in DMEM/0.5% FBS. (C) Overexpression of both hTNX-FGFFFM and ITGA11 did not cause induction of COL1A1 expression. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGFFFM (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGFFFM and ITGA11 (lane 6) in DMEM/0.5% FBS. After transfection followed by cell culture, cell lysate extraction and RNA purification, the expression levels of ACTA2, COL1A1 and TGFB1 were examined by reverse transcription-quantitative PCR. (A-C) The expression level of each gene in the control (lane 1, RNA from cells without transfection) was set to 1.0, and the relative expression level of each gene compared with that of the control is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1), one-way ANOVA with Dunnett's post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG); hTNX-FGFFFM, PRDCGEEMQNGAGAS peptide from hTNX-FG; hTNX-FGFFFL, QNGAGASRTSTIFL peptide from hTNX-FG; hTNX-FGpeptide2-5, GGLRIPF peptide from hTNX-FG.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Sequencing, Expressing, Over Expression, Transfection, Cell Culture, Extraction, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 in addition to inhibitors. (A) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 with a TGFBRI inhibitor (SB525334). DMSO (lanes 1, 2 and 3) and SB525334 (lane 3) were added to the culture medium (DMEM/0.5% FBS) after the transfection of expression plasmids for both hTNX-FGFFFF and ITGA11 in LX-2 cells (lanes 2 and 3). (B) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 with a YAP inhibitor (verteporfin). DMSO (lanes 1, 2 and 3) and vertepofin (lane 3) were added to the culture medium (DMEM/0.5% FBS) after the transfection of expression plasmids for both hTNX-FGFFFF and ITGA11 in LX-2 cells (lanes 2 and 3). Subsequently, the cells were cultured followed by cell lysate extraction, RNA purification and reverse transcription-quantitative PCR. (A and B) The expression level of each gene [ ACTA2, COL1A1 and TGFB1 for (A) and ACTA2, COL1A1 and YAP1 for (B)] in the control (lane 1) was set to 1.0, and the relative expression level of each gene compared with that of the control (lane 1) is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1); ## P<0.01 vs. lane 2, one-way ANOVA with the Bonferroni post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; YAP, Yes-associated protein; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG).

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Expressing, Over Expression, Transfection, Cell Culture, Extraction, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Effect of YAP1 knockdown on induction of COL1A1 expression. YAP1 was knocked down with YAP1 siRNA prior to overexpression of both hTNX-FGFFFF and ITGA11, and then the expression levels of ACTA2 and COL1A1 were analyzed by reverse transcription-quantitative PCR in LX-2 cells. RNA from cells treated with transfection of control siRNA only (lane 1), transfection of control siRNA followed by co-transfection with both hTNX-FGFFFF and ITGA11 expression plasmids (lane 2) and transfection of YAP1 siRNA followed by co-transfection with both hTNX-FGFFFF and ITGA11 expression plasmids (lane 3) was used. The expression level of each gene ( ACTA2, COL1A1 and YAP1 ) in the control (control siRNA only) (lane 1) was set to 1.0, and the relative expression level of each gene compared with that of the control (control siRNA) is shown (n=3). Data are presented as the mean ± SD. **P<0.01 vs. control (lane 1); # P<0.05, ## P<0.01 vs. lane 2, one-way ANOVA with the Bonferroni post hoc test. At the bottom of the figure, the relative expression levels of YAP1, hTNX-FGFFFF and ITGA11 are also shown, setting lane 2 to 1.0, since hTNX-FGFFFF expression was not detected in lane 1. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; YAP1, Yes-associated protein 1; siRNA, small interfering RNA; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG).

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Knockdown, Expressing, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Control, Cotransfection, Small Interfering RNA

Journal: PLoS ONE

Article Title: The Bi-Functional Organization of Human Basement Membranes

doi: 10.1371/journal.pone.0067660

Figure Lengend Snippet: The retinal surface (R) of the ILM and the endothelial (En) and epithelial (Ep) sides of DM and LC were stained with a polyclonal (red, LNp; A, C, D) and a monoclonal antibody to laminin (red, LNm, B), whereas the vitreal (V) or stromal side (St) of the BMs were labeled with an antibody to the 7S domain of collagen IV α3/4/5 (green; A-C, E). The asymmetry of BMs was also detected by single and double labeling of crossections of an isolated ILM (F-H) and an ILM in situ (I). The TEM micrographs in panel (K, L) show crossections of isolated ILMs stained for 7S collagen IV α3/4/5 (K) and laminin (L). The dark label shows the localization of 7S collagen IV on the vitreal side (K) and laminin on retinal side of the ILM (L). An asymmetric distribution for laminin and collagen 7S was also detected for the Descemet’s membranes (M, N) and the lens capsule (O, P). The sections were stained for laminin (red; M-P) and collagen IV 7S (green; N, P). Scale Bars: A-E: 100 µm; F-I and M-P: 10 µm; K, L, I: 1 µm.

Article Snippet: The blots were incubated with a polyclonal anti-collagen IV antibody (Rockland) followed by alkaline-phosphatase-conjugated goat anti-rabbit (1∶2000; Jackson ImmunoResearch, West grove, PA).

Techniques: Staining, Labeling, Isolation, In Situ

Journal: PLoS ONE

Article Title: The Bi-Functional Organization of Human Basement Membranes

doi: 10.1371/journal.pone.0067660

Figure Lengend Snippet: The domain specificity was determined by Western blotting (lanes 1–6, panel A) and immunoprecipitation (lanes 7, 8, panel A) using adult human lens capsules as sample. The western blot of lanes 1 and 2, stained with a polyclonal antiserum to human collagen IV, shows the complex peptide banding pattern of intact collagen IV from human lens capsules age 24 (lane 1) and age 65 (lane 2). Staining of the 65 year-old lens capsule sample with the J3-2 Mab (lane 3) shows a similar, but not entirely identical banding pattern. Red bars mark corresponding bands. The non-collagenous (NC1 and 7S) domains of collagen IV were detected in western blots after digestion of lens capsules with collagenase: western blots with the soluble supernatant from digested lens capsule samples, stained with polyclonal collagen IV antiserum, shows the NC-domains of collagen IV as a long smear at 300 kD and a ladder of lower molecular weight peptides (lane 4, panel A). The NC1 domain of α3 collagen IV appears as a sharp band below 49 kD (lane 5, red bar; panel A) as shown by staining with a monoclonal antibody specific to the collagen IV α3 NC1 domain. The high molecular weight smear stained by Mab J3-2 correspond to the variably crosslinked 7S domain (Lane 6, panel A). The smear is due to the glycosylation of the 7S domain , . For immunoprecipitation, the collagenase-digest of lens capsule was incubated with Mab J3-2 or anti-laminin as a control followed by anti-mouse IgM or anti-rabbit IgG agarose. The beads were washed and the bound peptide released by boiling in high molar urea/SDS sample buffer. The released peptides were separated by SDS PAGE and western blotted. The immuno-precipitated peptides were visualized in the blot by a polyclonal anti-collagen IV antibody/alkaline phosphatase-conjugated secondary antibody. For the J3-2 pull-down, a smear of approximately 300 kD was detected (lane 7, panel A), similar to the smear detected by Western blotting (lane 6). A control pull-down with anti-laminin is shown in lane 8. The blots shows that Mab J3-2 i) recognizes human collagen IV, that it ii) recognizes an NC domain of collagen IV and that iii) the molecular weight of the detected peptides are different from the molecular weight of the NC1 domain but identical to the molecular weight of the 7S domain of collagen IV from glomerular BM and lens capsule. To determine the chain-specificity of the antibody, sections of adult human retina were stained with Mab J3-2 (B), to the NC1 domain of collagen IV α3 (C), to the NC1 domain of collagen IV α1 (D) and to the NC1 domain of collagen α5 (E). A3, α4 (not shown) and α5 collagen IV are very prominent in the ILM but sparse in the BMs of the retinal blood vessels (C, E), whereas α1/2 was very sparse in the ILM but abundant in the blood vessels (D). The staining with Mab J3-2 (A) resembles most closely the staining of collagen α3 α4 and α5, and it is very different from the distribution of collagen IV α1/α2. Scale Bar: 50 µm.

Article Snippet: The blots were incubated with a polyclonal anti-collagen IV antibody (Rockland) followed by alkaline-phosphatase-conjugated goat anti-rabbit (1∶2000; Jackson ImmunoResearch, West grove, PA).

Techniques: Western Blot, Immunoprecipitation, Capsules, Staining, Molecular Weight, High Molecular Weight, Glycoproteomics, Incubation, Control, SDS Page

Journal: PLoS ONE

Article Title: The Bi-Functional Organization of Human Basement Membranes

doi: 10.1371/journal.pone.0067660

Figure Lengend Snippet: Staining with polyclonal antibodies resulted in a uniform and even labeling of the retinal (R) and vitreal (V) side of the ILM. In contrast, staining of ILM with antibodies specific to the 7S (B) or the NC1 domain (C) of collagen IV alpha3/4/5 resulted in the selective labeling of the vitreal (B) or the retinal side (C). The distribution of laminin in the ILM (D) is very similar to that of collagen IV NC1 at the epithelial side. The ILM sample in panel C and D was double-labeled; the NC1 domain was labeled with a Cy3 (red; C) secondary antibody, whereas laminin was detected with a Cy2 (green; D) secondary antibody. Scale Bar: 100 µm.

Article Snippet: The blots were incubated with a polyclonal anti-collagen IV antibody (Rockland) followed by alkaline-phosphatase-conjugated goat anti-rabbit (1∶2000; Jackson ImmunoResearch, West grove, PA).

Techniques: Staining, Labeling