Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 1. Biological activity of hLMP2 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#2 (T type) clone and flat revertants of the SKN-LMP2#122 (F type) clone (magnification 100). Changes in human uterine LMS cell line, SKN-transfectants, SKN-CEM9 (T type) clone, and SKN-LMP2wt (F type) clone xenograft volumes in mice (n = 8). Representative photographs of xenografts in mice (Left). Tumor growth of SKN-LMP2 was markedly reduced in comparison with that of the control transfectant SKN-CEM9 (T type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-CEM9 (T type) clone and SKN-LMP2 (F type) clone (Right). (B) RT-PCR experiments revealed hLMP2, hLMP7, Calponin h1, SRF, cyclin B and b-actin mRNA expression in tumors. Precursor LMP2 or LMP7 (pre-LMP2, pre- LMP7) and mature LMP2 or LMP7 (LMP2, LMP7) are shown. (C) Western blotting revealed LMP2, LMP7, calponin h1, SRF, cyclin B, and b-actin in SKN-transfectant clones. (D) The luciferase reporter vectors containing the hCalponin h1 promoter with wild type SRF binding sites (Calponin-wt-Luc.), mutant SRF binding sites (Calponin-mut-Luc.), or empty luciferase reporter vector (Basic-Luc.) [23] were transiently co-transfected with pSV-b-galactosidase in SKN-transfectants, SKN-CEM9#2, SKN-LMP2#121, or SKN- LMP2#122 clones for the final 48 h, and then luciferase activities were measured. Values were normalized to those obtained with the co-transfected pSV-b-galactosidase expression vector. Each assay was performed at least three times and in triplicate. Luciferase reporter assays showed that LMP2 expression markedly induced calponin h1 promoter activation. Data are presented as the mean from three independent experiments (⁄S.D.). The experiments were performed four times with similar results. SKN transformantsa, CEM9 SKN-CEM9#2; LMP2, SKN-LMP2wt#121, SKN-LMP2wt#122. Detail is shown in SFig. 2, SFig. 3 and STable 3. RT-PCRb, total RNA samples were isolated from the individual xenografted-tumors, which were removed at 5 weeks after xenografting. W.B.c, W.B. are performed with the total cell lysates from SKN transformants.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, Comparison, Control, Transfection, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Clone Assay, Luciferase, Binding Assay, Mutagenesis, Plasmid Preparation, Activation Assay, Isolation

Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 2. Biological activity of calponin h1 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#1Scr.shRNA (T type) clone, SKN-CEM9#2 calponin h1shRNA (T type) clone, SKN-CEM9#2 (T type) clone, SKN-LMP2#1Scr.shRNA (F type) clone, and SKN-LMP2#2Calponin h1shRNA (T type) clone of the SKN-LMP2 (F type) clone (magnification 60). The growth rates of the SKN-transfectant clones were measured as population doubling time (PDT). (B) Western blotting and RT-PCR experiments revealed calponin h1, precursor LMP2 (pre-LMP2), mature LMP2 (LMP2), and b-actin in SKN-transfectant clones. SKN transformantsa, CEM9#3 Scr.shRNA, CEM9#4 Calponin h1shRNA, LMP2#1 Scr.shRNA, LMP2#2 Calponin h1shRNA, Detail is shown in Table 1 and SFig. 5 and STable 3. (C) Changes in the human uterine LMS cell line, SKN-transfectant, SKN-CEM9#2 (T type) clone, SKN-LMP2wt#2/Calponin h1shRNA (T type) clone, and SKN-LMP2wt#1/ Scr.shRNA (F type) clone xenograft volumes in mice (n = 3). Representative photographs of xenografts in mice (Left). Tumor growth of the SKN-LMP2wt#2/Calponin h1shRNA (T type) clone is mildly increased in comparison with that of the SKN-LMP2wt#1/Scr.shRNA (F type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-transfectant clones (Right). RT-PCR experiments revealed hCalponin h1, hLMP2 and b-actin mRNA expression in tumors (Bottom). Experiments were performed three times with similar results. SKN-CEM9c, SKN- CEM9#2; LMP2wt+Calponin h1shRNAd, SKN-LMP2wt#2/ CalponinshRNA; LMP2wt/Scr.shRNAe, SKN-LMP2wt#1/Scr.shRNA. Details of SKN transfectants are shown in Table 1, SFig. 5 and STable 3. RT-PCRf, total RNA samples were isolated from the individual xenografted-tumors, which were removed from BALB/c nu/numice at 5 weeks after xenografting. Xenograftsg, BALB/c nu/nu mice were inoculated with SKN-CEM9#2, SKN-LMP2wt#2/CalponinshRNA or SKN-LMP2wt#1/Scr.shRNA.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, shRNA, Transfection, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, Injection, Expressing, Isolation

Journal: Oncotarget

Article Title: Mitochondrial fission induces glycolytic reprogramming in cancer-associated myofibroblasts, driving stromal lactate production, and early tumor growth.

doi: 10.18632/oncotarget.574

Figure Lengend Snippet: Figure 6: Fibroblasts over-expressing MFF show myofibroblastic features. To evaluate if MFF-fibroblasts acquire myofibroblastic features, cells were analyzed by immuno-blotting with antibodies directed against α-SMA and calponin. Note that MFF-fibroblasts show increased expression of two myo-fibroblast markers, namely α-SMA and calponin, indicating that MFF promotes a myofibroblastic differentiation. β-actin was used as an equal protein loading control.

Article Snippet: The following antibodies were used: MFF (Abcam, ab81127); MCT4 (Sigma-Aldrich, SAB4503555); Smooth Muscle Actin (Dako, M0851); Calponin 1/2/3 (FL-297) (Santa Cruz, sc-28545); BNIP3 (Abcam, ab10433); BNIP3L (Abcam, ab8399); Cathepsin B (FL-339) (Santa Cruz, sc-13985); LC3 (Abcam, ab48395); NF-kB p65 (Cell Signaling, 3034); phospho-NF-kB p65 (Cell Signaling, 3037); Mitoprofile total OXPHOS cocktail (Mitosciences, MS601).

Techniques: Expressing, Control

Journal: Bioactive Materials

Article Title: A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: Enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway

doi: 10.1016/j.bioactmat.2020.08.018

Figure Lengend Snippet: RNA genome sequencing analysis of contractile and synthetic PCASMCs with or without ATO treating for 8 h, and qPCR validation of genes and signals. (A) The schematic diagram for the phenotype induction and ATO treating of PCSMCs. (B) Venn diagram. (C) Gene Ontology (GO) enrichment analysis. (D) and (E) are the statistical tables of changes in ATO treatment, related cell processes (D) and main gene expression (E) of synthetic PCASMCs (SH2 vs SH0). (F) qPCR of α-SMA, Calponin, RhoA, ROCK and YAP for A7r5. SS0: contractile PCASMCs without ATO treatment; SS2: contractile PCASMCs treated with 2 μM ATO for 8 h; SH0: synthetic PCASMCs without ATO treatment; and SH2: synthetic PCASMCs treated with 2 μM ATO for 8 h. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μM of ATO, respectively.

Article Snippet: The following primary antibodies against α-SMA (ab7817, Abcam, 1:300), SM22α (ab14106, Abcam, 1:300), Calponin (bs-0095R, Bioss, 1:250), YAP (D8H1X, 14074, Cell signal technology, 1:300), p-YAP (ab62751, Abcam, 1:300), and phalloidin (Proteintech, 1:200) were used.

Techniques: Sequencing, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Sp1 Plays an Important Role in Vascular Calcification Both In Vivo and In Vitro

doi: 10.1161/JAHA.117.007555

Figure Lengend Snippet: Sp1 gene silencing prevented VSMC transdifferentiation into osteoblast‐like cells. VSMC s were infected with adenovirus harboring Sp1 gene (Ad‐Sp1) or empty virus (Vehicle). A, Immunofluorescence staining and quantification of Sp1 expression in VSMC s (n=4, Scale bar: 20 μm). Representative Western blot images and quantification of (B) BMP 2, (C) Runx2, (D) α‐ SMA , (E) calponin expression in treated primary rat VSMC s, n=5. Data are mean± SEM . * P <0.05, β‐ GP or β‐ GP vs Control; # P <0.05 β‐ GP +Ad‐Sp1 vs β‐ GP +Vehicle and β‐ GP +Ad‐Sp1 vs β‐ GP +Vehicle. Ad‐Sp1 indicates adenovirus harboring Sp1 knockdown gene; BMP 2, bone morphogenetic protein 2; CON, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; β‐GP, β‐glycerophosphate; Runx2, run‐related transcription factor 2; α‐ SMA , α‐smooth muscle actin; VDN, vitamin D3 and nicotine induced calcification; VSMC s, vascular smooth muscle cells.

Article Snippet: Primary antibodies against Sp1 (0.1 μg/mL, Millipore), BMP2 (1:1000 dilution), calponin (1:2000 dilution) (Proteintech Group, Chicago, IL), Runx2 (1:1000 dilution), α‐SMA (1:1000 dilution), caspase‐3 (1:1000 dilution), β‐actin (1:1000 dilution), GAPDH (1:1000 dilution) (Cell Signaling Technology), and horseradish peroxidase‐conjugated secondary antibody (Beijing Zhong Shan Golden Bridge Biological Technology, China) were used.

Techniques: Infection, Virus, Immunofluorescence, Staining, Expressing, Western Blot, Control, Knockdown

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Sp1 Plays an Important Role in Vascular Calcification Both In Vivo and In Vitro

doi: 10.1161/JAHA.117.007555

Figure Lengend Snippet: Sp1 inhibitor mithramycin suppressed β‐ GP ‐induced phenotype switching of VSMC s. VSMC s were treated with β‐ GP and mithramycin in different doses. A, Representative Western blot bands and (B and C) quantification of BMP 2, Runx2, α‐ SMA , and calponin protein expression. D and E, Real‐time quantitative PCR analyses of BMP 2, Runx2, α‐ SMA , and calponin mRNA expression. Data are mean± SEM . n=4, * P <0.05 β‐ GP vs Control; # P <0.05 β‐ GP +mithramycin vs β‐ GP . BMP 2 indicates bone morphogenetic protein 2; β‐ GP , β‐glycerophosphate; MTM , mithramycin; PCR, polymerase chain reaction; Runx2, run‐related transcription factor 2; α‐ SMA , α‐smooth muscle actin; Sp1, specific protein 1; VSMC s, vascular smooth muscle cells.

Article Snippet: Primary antibodies against Sp1 (0.1 μg/mL, Millipore), BMP2 (1:1000 dilution), calponin (1:2000 dilution) (Proteintech Group, Chicago, IL), Runx2 (1:1000 dilution), α‐SMA (1:1000 dilution), caspase‐3 (1:1000 dilution), β‐actin (1:1000 dilution), GAPDH (1:1000 dilution) (Cell Signaling Technology), and horseradish peroxidase‐conjugated secondary antibody (Beijing Zhong Shan Golden Bridge Biological Technology, China) were used.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Control, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Sp1 Plays an Important Role in Vascular Calcification Both In Vivo and In Vitro

doi: 10.1161/JAHA.117.007555

Figure Lengend Snippet: Sp1 gene silencing alleviated vascular calcification in aorta and inhibited VSMC s phenotype switching. Rats were injected with either adenovirus harboring Sp1 gene (Ad‐Sp1) or empty virus (Vehicle). A and B, Western blot analysis of Sp1 in rat aorta. C, Alizarin red staining of aorta ( pH 4.2) (Scale bar: 50 μm). D, Quantitative analysis of calcium deposition in aorta normalized to protein content. E, Alkaline phosphatase ( ALP ) activity was measured and normalized to protein content for quantitative analysis. F through I, Representative Western blot bands and quantification of BMP 2, Runx2, α‐ SMA , and calponin expression in aorta extracts. Data are mean± SEM . n=15 aortic sections from 5 rats in each group, * P <0.05β‐ GP or VDN vs Control; # P <0.05 β‐ GP +Ad‐Sp1 vs β‐ GP +Vehicle and VDN +Ad‐Sp1 vs VDN +Vehicle. Ad‐Sp1 indicates adenovirus harboring Sp1 knockdown gene; ( ALP ), alkaline phosphatase; BMP 2, bone morphogenetic protein 2; CON, control; α‐ SMA , α‐smooth muscle actin; VDN, vitamin D3 and nicotine–induced calcification; VSMC s, vascular smooth muscle cells.

Article Snippet: Primary antibodies against Sp1 (0.1 μg/mL, Millipore), BMP2 (1:1000 dilution), calponin (1:2000 dilution) (Proteintech Group, Chicago, IL), Runx2 (1:1000 dilution), α‐SMA (1:1000 dilution), caspase‐3 (1:1000 dilution), β‐actin (1:1000 dilution), GAPDH (1:1000 dilution) (Cell Signaling Technology), and horseradish peroxidase‐conjugated secondary antibody (Beijing Zhong Shan Golden Bridge Biological Technology, China) were used.

Techniques: Injection, Virus, Western Blot, Staining, Activity Assay, Expressing, Control, Knockdown