Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-335-5p regulates the proliferation, migration and phenotypic switching of vascular smooth muscle cells in aortic dissection by directly regulating SP1

doi: 10.3724/abbs.2022081

Figure Lengend Snippet: SP1 is involved in HA-VSMC proliferation, migration and phenotypic switching (A) The transfection efficiency of SP1/pGPH1 was examined by RT-qPCR ( n=3). (B) The proliferation of HA-VSMCs in the different groups at different times was detected by the CCK-8 assay ( n=3). (C) Representative images of Transwell assay of the different groups (upper, scale bar =50 μm), and quantitative analysis of the number of migrated cells (lower, n=3). (D) Representative images of the wound-healing assay of the different groups (left, scale bar =200 μm) are shown, and the wound closure ratio was analyzed (right, n=3). (E) RT-qPCR analysis of the mRNA expression levels of contractile markers (α-SMA, SM22α, and CNN1) and synthetic markers (OPN and Collagen I) in the different groups ( n=3). Data are expressed as the mean±SD based on triplicate independent experiments and were analyzed by one-way ANOVA. * P<0.05, ** P<0.01 and *** P<0.001.

Article Snippet: The membranes were blocked with 5% nonfat milk (P0216; Beyotime) for 2 h at room temperature and then incubated with primary antibodies against α-SMA (1:1000, bs-10196R; Bioss, Beijing, China), SM22α (1:1000, 10493-1-AP; ProteinTech, Rosemont, USA), CNN1 (1:1000, bs-0095R; Bioss), OPN (1:1000, bs-0026R; Bioss), Collagen I (1:1000, 14695-1-AP; ProteinTech) and β-actin (1:2000, 20536-1-AP; ProteinTech) overnight at 4°C.

Techniques: Migration, Transfection, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Wound Healing Assay, Expressing

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: Cell reports

Article Title: Sensing Actin Dynamics through Adherens Junctions

doi: 10.1016/j.celrep.2020.01.106

Figure Lengend Snippet: (A–D) Projections of all x-y optical slices of A431 cells stained for (A) E-cadherin (Ecad, red) and F-actin (actin, green); (B) Ecad (green), F-actin (red), and myosin IIA (myosin II, blue); (C) Ecad (green), actin, and calponin-3 (Cnn3); and (D) Cnn3 (green) and myosin IIA (red).

Article Snippet: The plasmid encoding PA-mCH-tagged b-actin was a gift from Dr. Verkhusha (Addgene, plasmid # 31949). pRc-CNN3-mGFP plasmid was generated by using Myc-DDK tagged CNN3 (Origene, RC200488).

Techniques: Staining

Journal: Cell reports

Article Title: Sensing Actin Dynamics through Adherens Junctions

doi: 10.1016/j.celrep.2020.01.106

Figure Lengend Snippet: (A–D) Projections of all x-y optical slices of Cnn3-Dn-expressing A431 cells imaged for Dendra2 fluorescence (green, Cnn3-Dn) and stained for (A) Ecad, (B) actin, (C) vinculin (Vcn), and (D) mena. Scale bar, 10 μm. The zoomed areas (indicated by dashed boxes in Cnn3-Dn images) are presented at the bottom. Scale bar, 5 μm. Arrowheads indicate the gaps in Cnn3-Dn distribution along pAJ-linked bundles that are precisely matched by the antibody staining.

Article Snippet: The plasmid encoding PA-mCH-tagged b-actin was a gift from Dr. Verkhusha (Addgene, plasmid # 31949). pRc-CNN3-mGFP plasmid was generated by using Myc-DDK tagged CNN3 (Origene, RC200488).

Techniques: Expressing, Fluorescence, Staining

Journal: Cell reports

Article Title: Sensing Actin Dynamics through Adherens Junctions

doi: 10.1016/j.celrep.2020.01.106

Figure Lengend Snippet: (A–C) Maximum projection view of control A431-EcGFP cells (A431-EcGFP) or after Cfl1-KO (A431-EcGFP-Cfl1-KO). Cells were imaged for GFP (EcGFP) and for (A) Cfl1 (Cfl, red), (B) actin (actin, red), and (C) Cnn-3 (Cnn3, red). Scale bar, 10 μm. The zoomed areas (indicated by dashed boxes in EcGFP images) are presented in the insets (A) or at the bottom (B and C). Scale bar, 5 μm.

Article Snippet: The plasmid encoding PA-mCH-tagged b-actin was a gift from Dr. Verkhusha (Addgene, plasmid # 31949). pRc-CNN3-mGFP plasmid was generated by using Myc-DDK tagged CNN3 (Origene, RC200488).

Techniques:

Journal: Cell reports

Article Title: Sensing Actin Dynamics through Adherens Junctions

doi: 10.1016/j.celrep.2020.01.106

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The plasmid encoding PA-mCH-tagged b-actin was a gift from Dr. Verkhusha (Addgene, plasmid # 31949). pRc-CNN3-mGFP plasmid was generated by using Myc-DDK tagged CNN3 (Origene, RC200488).

Techniques: Recombinant, CRISPR, Plasmid Preparation, Software

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