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: Molecular Medicine Reports

Article Title: Rapamycin inhibits CaCl 2 -induced thoracic aortic aneurysm formation in rats through mTOR-mediated suppression of proinflammatory mediators

doi: 10.3892/mmr.2017.6844

Figure Lengend Snippet: Antibodies and dilutions.

Article Snippet: Calponin , sc70487 , Santa cruz , Mouse , 1:100.

Techniques:

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Effects of inhibition of AGEs on coronary artery tensions and BK channel densities and protein expression (a) Representative tracings for 60 mmol/L KCl and 100 nmol/L IBTX induced vascular tension alterations of coronary arterial rings from C+V, DM+V, C+A and DM+A groups. (b) Graph data showing the vascular tension alterations induced by KCl. (c) Graph data showing the vascular tension alterations (IBTX/KCl). (d and e) Whole-cell potassium currents before and after application of 100 nmol/L IBTX, and the I-V relationship of IBTX-sensitive currents of control and AGEs-cultured freshly isolated rat coronary arterial SMCs ( n = 3∼6 per group). (f) The representative tracings of baseline potassium currents and potassium currents after application of 100 nM IBTX in rat coronary arterial SMCs of the C+V, DM+V, C+A and DM+A groups, respectively ( n = 3∼5 per group). (g) Graph data showing IBTX-sensitive current densities at the testing potential of +100 mV in rat coronary arterial SMCs of the four groups. (h–j) The protein expressions of BK-α and BK-β1 in human coronary arterial SMCs in the BSA and BSA-AGEs groups ( n = 6∼9 per group). Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels. (k-l) The mRNA expression of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. β-actin was used as an internal control to normalize differences in the amount of total RNA in each rat sample ( n = 4 per group). (m and n) The mRNA expression of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. GAPDH was used as an internal control to normalize differences in the amount of total RNA in each cell sample ( n = 4∼5 per group). (o–q) Protein expressions of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5 per group). (r–t) Protein expressions of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5∼9 per group). (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine).

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Inhibition, Expressing, Control, Cell Culture, Isolation

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of Akt in AGEs-mediated FBXO32-induced BK-β1 degradation (a and b) Protein expression of FBXO32 in rat coronary arteries of four groups ( n = 5 per group). (c and d) Protein expression of FBXO32 in human coronary arterial SMCs of four cell groups. Quantitative analysis of FBXO32 was normalized to GAPDH protein expression levels. (e–g) Phosphorylation levels of Akt and total Akt in rat coronary arteries of four groups ( n = 8 per group). (h–j) Phosphorylation levels of Akt and total Akt in human coronary arterial SMCs of four groups ( n = 3 per group). The phosphorylation level of Akt (k and n) and the protein expressions of FBXO32 (l and o) and BK-β1 (m and p) were measured after human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose with aminoguanidine in the absence or presence of MK2206 (0.3 μM) ( n = 5∼10 per group). MK2206 was added at the beginning and remained for 6 h (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine.)

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Expressing, Phospho-proteomics, Incubation, Control

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of AMPK in Akt-mediated FBXO32-induced BK-β1 degradation by AGEs (a–c) Protein expression of p-AMPK and AMPK in rat coronary arteries from the four groups ( n = 8 per group). (d–f) Protein expression of p-AMPK and AMPK in human coronary arterial SMCs from the four groups ( n = 9 per group). Quantitative analysis of p-AMPK and AMPK was normalized to GAPDH protein expression levels. (g) Human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose and aminoguanidine in the absence or presence of Compound C (CC, 1 μM). Subsequently, the phosphorylation level of AMPK (h and i), AKT (j and k), and the protein expressions of FBXO32 (l) and BK-β1 (m) were measured ( n = 8 and 9 per group). Quantitative analysis of FBXO32 and BK-β1 was normalized to GAPDH protein expression levels.

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Expressing, Incubation, Phospho-proteomics

Journal: The Journal of allergy and clinical immunology

Article Title: A functional splicing variant associated with decreased asthma risk abolishes the ability of gasdermin B ( GSMDB ) to induce epithelial cell pyroptosis

doi: 10.1016/j.jaci.2017.11.040

Figure Lengend Snippet: A) Relative GSDMB mRNA expression in human primary airway smooth muscle (ASM), lung fibroblasts (FIB), and normal human bronchial epithelial (NHBE) cells over the course of air-liquid interface (ALI) culture. ACTB (β-actin) was used as an internal control. B) Relative GSDMB mRNA expression in sorted β−tubulin IV-positive ciliated NHBE and MUC5AC-postive goblet NHBE cells. Graphs show mean of fold-change from three different donors; n=3, +/− SEM. C and D) Immunostaining of GSDMB protein in NHBE (normal human bronchial epithelial) cells at day 21 of ALI culture (C) and lung tissue (D) shows GSDMB expression in ciliated cells. Ciliated cells were stained for β−tubulin IV and nuclei were visualized by DAPI staining.

Article Snippet: Primary human airway smooth muscle (ASM) cells and normal human lung fibroblasts were obtained from Lonza.

Techniques: Expressing, Control, Immunostaining, Staining