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: Nature medicine

Article Title: Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.

doi: 10.1038/nm.3901

Figure Lengend Snippet: Figure 2 Renal epithelial cells undergo a partial EMT after UUO. (a) Ksp1.3-Cre;tdTomatofl/fl mice were subjected to UUO and kidneys were collected 15 ds after surgery. Representative images (n = 20) of sections of kidneys from Ksp1.3-Cre;tdTomatofl/fl mice (KCT) 15 d after being subjected to UUO. Images are for obstructed (KCT + UUO) and contralateral non-obstructed kidneys (Control) from KCT mice stained for LTA (Lotus tetragonolobus agglutinin) + PNA (peanut agglutinin) (markers of proximal and distal tubules17), α-SMA and DAPI. tdTomato fluorescent protein expression was directly visualized. Asterisks (*) indicate α-SMA expression in tubular cells that have lost lectin expression 15 d after UUO. dt, dilated tubule; g, glomerulus; t, tubule. (b) Number of dilated and total tubules in kidneys from WT and SFKC mice 15 d after UUO. Ten different fields were counted in 12 mice per group. Data represent mean ± s.e.m. *P < 0.05, NS, nonsignificant; Mann-Whitney test. (c) Representative images (n = 5) of double Snail1 and α-SMA immunolabeling in sections from obstructed kidneys of WT mice euthanized 15 d after surgery. White asterisks indicate Snail1-positive nuclei. Green asterisks indicate double-positive Snail1 and α-SMA tubular cells. (d) Representative images (n = 5) of lectin (LTA) and Snail1 immunofluorescence in sections from the same kidneys used in b. Scale bars, 50 µm.

Article Snippet: The sections were incubated overnight at 4 °C with the following primary antibodies: biotinylated Lotus tetragonolobus agglutinin, LTA (B-1325, Vector Laboratories, Burlingame, CA); biotinylated peanut agglutinin, PNA (B-1075, Vector Laboratories, Burlingame, CA); α smooth muscle actin, (α-SMA) (Sigma-Aldrich, St Louis, MO); vimentin (sc-7557, Santa Cruz Biotechnology, Santa Cruz, CA); Snail1 (Ab1773; Abcam PLC, Cambridge, MA); phosphoNF-κB p65 (Ser276) (3037S, Cell Signaling Technology, Inc., Danvers, MA); phospho-Smad2 (Ser465/467) (3101S, Cell Signaling Technology, Inc., Danvers, MA); F4/80 (MCA497GA, Serotec, Oxford,UK); estrogen receptor alpha, ER-α (sc-543, Santa Cruz Biotechnology, Santa Cruz, CA); CD163 (sc-18796 Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Staining, Expressing, MANN-WHITNEY, Immunolabeling, Immunofluorescence

Journal: Nature medicine

Article Title: Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.

doi: 10.1038/nm.3901

Figure Lengend Snippet: Figure 4 Snail1 reactivation is required for the development of folic acid-induced fibrosis. FA was intraperitoneally injected to WT or SFKC mice, which were euthanized 34 d after FA administration. (a) Representative images (n = 5) of H&E and Sirius red stainings, as well as immunohistochemical analysis of LTA, PNA, α-SMA, F4/80 and CD163 on sections from WT and SFKC mice treated with 300 mM NaHCO3 vehicle (control) or FA. Tissue sections are representative of five mice per group. (b) Snai1, Col1a1, Cadh1, Cadh16, Vim, Acta2, Tgfb1, Tnf, Ccl2 and Ccl5 mRNA levels determined by qRT-PCR in vehicle (Control) or FA-treated WT and SFKC mice. Data are normalized to vehicle-treated WT levels and represent mean ± s.e.m. for groups of four mice. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test. (c) Kidney lysates from WT or SFKC mice treated with FA were incubated with a cytokine array. Data represent the signals detected for specific cytokines in SFKC lysates relative to those in WT lysates. Error bars represent mean ± s.e.m. (d) Sirius red staining of sections from kidneys from WT or SFKC mice treated with FA was quantified and represented relative to vehicle-treated mice in three mice per group. ****P < 0.0001; Mann-Whitney test. Error bars show means ± s.e.m. Scale bars, 50 µm.

Article Snippet: The sections were incubated overnight at 4 °C with the following primary antibodies: biotinylated Lotus tetragonolobus agglutinin, LTA (B-1325, Vector Laboratories, Burlingame, CA); biotinylated peanut agglutinin, PNA (B-1075, Vector Laboratories, Burlingame, CA); α smooth muscle actin, (α-SMA) (Sigma-Aldrich, St Louis, MO); vimentin (sc-7557, Santa Cruz Biotechnology, Santa Cruz, CA); Snail1 (Ab1773; Abcam PLC, Cambridge, MA); phosphoNF-κB p65 (Ser276) (3037S, Cell Signaling Technology, Inc., Danvers, MA); phospho-Smad2 (Ser465/467) (3101S, Cell Signaling Technology, Inc., Danvers, MA); F4/80 (MCA497GA, Serotec, Oxford,UK); estrogen receptor alpha, ER-α (sc-543, Santa Cruz Biotechnology, Santa Cruz, CA); CD163 (sc-18796 Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Injection, Immunohistochemical staining, Control, Quantitative RT-PCR, Comparison, Incubation, Staining, MANN-WHITNEY

Journal: Nature medicine

Article Title: Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.

doi: 10.1038/nm.3901

Figure Lengend Snippet: Figure 5 Snail1-induced fibrosis can be reversed in vivo. (a) Scheme of the experimental approach. Mice were treated with (1) vehicle (corn oil, −TAM); (2) tamoxifen for 8 weeks (+TAM); (3) tamoxifen for 8 weeks followed by vehicle treatment for another 8 weeks (+TAM −TAM). (b) Anti-human estrogen receptor (hER) immunohistochemical analysis showing exogenous Snail1 protein expression and activation upon tamoxifen treatment. (c) Representative images (n = 6 from six mice per group) of H&E and Sirius red staining, and immunohistochemical analysis of α-smooth muscle actin (α-SMA), vimentin, phospho-NF- κB (pNF-κB), F4/80 and CD163 from kidney sections from mice in a. (d) mRNA levels detected by qRT-PCR in kidneys from Snail1- ERT2 mice showing Snai2, Vim, Tgfb1 Cadh1 and Cadh16 abundance upon Snail1 activation (2; +TAM) and deactivation after 8 weeks of tamoxifen removal (3; +TAM −TAM). Data are normalized to vehicle-treated kidney levels (1; −TAM) and represent mean ± s.e.m. for groups of four mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test. (e) Morphometric analysis of cortical interstitial fibrosis by quantification of Sirius red staining. Data are shown relative to vehicle treated controls (4 mice per group). **P < 0.01, ***P < 0.001, **** P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test. Error bars show means ± s.e.m. Scale bars, 50 µm.

Article Snippet: The sections were incubated overnight at 4 °C with the following primary antibodies: biotinylated Lotus tetragonolobus agglutinin, LTA (B-1325, Vector Laboratories, Burlingame, CA); biotinylated peanut agglutinin, PNA (B-1075, Vector Laboratories, Burlingame, CA); α smooth muscle actin, (α-SMA) (Sigma-Aldrich, St Louis, MO); vimentin (sc-7557, Santa Cruz Biotechnology, Santa Cruz, CA); Snail1 (Ab1773; Abcam PLC, Cambridge, MA); phosphoNF-κB p65 (Ser276) (3037S, Cell Signaling Technology, Inc., Danvers, MA); phospho-Smad2 (Ser465/467) (3101S, Cell Signaling Technology, Inc., Danvers, MA); F4/80 (MCA497GA, Serotec, Oxford,UK); estrogen receptor alpha, ER-α (sc-543, Santa Cruz Biotechnology, Santa Cruz, CA); CD163 (sc-18796 Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: In Vivo, Immunohistochemical staining, Expressing, Activation Assay, Staining, Quantitative RT-PCR, Comparison

Journal: Nature medicine

Article Title: Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.

doi: 10.1038/nm.3901

Figure Lengend Snippet: Figure 6 Snail1 inactivation reverts UUO- induced fibrosis in mice. (a) Scheme of the experimental approach. Seven days after UUO, mice were injected with vivo-morpholino (VI-MO) control (Control-MO) or Snail1-MOs (Snail1-MO1 or Snail1-MO2; Supplementary Fig. 9) every other day. Shown are the results obtained in mouse #5, representative of those in which the MO efficiently prevented Snail1 normal splicing. (b) Representative images (n = 5) of H&E and Sirius red staining, and immunohistochemical analysis of mesenchymal (vimentin and α-smooth muscle actin (α-SMA)) and inflammatory response markers (F4/80 and phospho-NFkB (pNFκB)) in kidney sections from mice in a. (c) Snai1, Snai2, Vim, Tgfb1, Cadh1 and Cadh16 mRNA levels detected by qRT-PCR in obstructed kidneys from WT mice treated with Snail1-MO. Data are normalized to contralateral non-obstructed kidney levels (NL) and represent mean ± s.e.m. for triplicates of data from a representative mouse in which Snail1-MO efficiently induced exon-skipping and Snai1 downregulation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA followed by Tukey’s multiple comparison test. (d) Sirius red quantification in the renal cortex of mice injected with Control-MO (average values from four mice) or Snail1-MO (mouse #5). ****P < 0.0001; Mann-Whitney test. Error bars show means ± s.e.m. Scale bars, 50 µm.

Article Snippet: The sections were incubated overnight at 4 °C with the following primary antibodies: biotinylated Lotus tetragonolobus agglutinin, LTA (B-1325, Vector Laboratories, Burlingame, CA); biotinylated peanut agglutinin, PNA (B-1075, Vector Laboratories, Burlingame, CA); α smooth muscle actin, (α-SMA) (Sigma-Aldrich, St Louis, MO); vimentin (sc-7557, Santa Cruz Biotechnology, Santa Cruz, CA); Snail1 (Ab1773; Abcam PLC, Cambridge, MA); phosphoNF-κB p65 (Ser276) (3037S, Cell Signaling Technology, Inc., Danvers, MA); phospho-Smad2 (Ser465/467) (3101S, Cell Signaling Technology, Inc., Danvers, MA); F4/80 (MCA497GA, Serotec, Oxford,UK); estrogen receptor alpha, ER-α (sc-543, Santa Cruz Biotechnology, Santa Cruz, CA); CD163 (sc-18796 Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Injection, Control, Staining, Immunohistochemical staining, Quantitative RT-PCR, Comparison, MANN-WHITNEY

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Adipose Mesenchymal Cells-Derived EVs Alleviate DOCA-Salt-Induced Hypertension by Promoting Cardio-Renal Protection

doi: 10.1016/j.omtm.2019.11.002

Figure Lengend Snippet: ASC-EV Treatment Promoted Important Attenuation of Kidney Fibrosis in the DOCA-Salt Model (A) Representative photomicrographs of kidney slices stained with Picrosirius red on 9-week-treated rats from the 4 experimental groups (scale bars, 50 μm). (B) α-SMA expression in the kidney of 9-week-treated rats under the different experimental conditions was determined by immunohistochemistry (n = 4 for each treatment) (scale bars, 50 μm). (C) Quantification of Picrosirius red stained area in kidney sections expressed as a percentage of the total area of the kidney, in 9-week-treated rats, measured for all the animals tested in the 4 experimental groups (SHAM, n = 4; UNX, n = 9; DOCA, n = 13; DOCA-EV, n = 10). (D−F) Western blot analysis of α-SMA and Desmin in the cortex of 9-week-treated UNX (n = 6), DOCA, and DOCA-EV (n = 8) rats. GAPDH was used as loading control. Values represent the fold increase with respect to UNX (considered as the reference = 1). (G−I) Analysis of COL1A1 , COL4A1 , and FN mRNA levels in the renal tissue in control SHAM (n = 3) and in all the uninephrectomized animal groups (UNX, n = 6; DOCA and DOCA-EV, n = 7). qRT-PCR data were expressed as RQ and UNX was used as the reference sample. Data represent mean ± SE. *indicates statistical difference to SHAM group, # indicates statistical difference to UNX group, and § indicates statistical difference to DOCA group; p < 0.05.

Article Snippet: The following primary antibodies were used: GAPDH, sc-32233 (1:1,000, Santa Cruz); α-SMA, MAB1420 (1:1,000, R&D Systems, Minneapolis, MN, USA); Desmin, ab32362 (1:1,000, Abcam); B cell lymphoma 2 (Bcl2), 2876 (1:1,000, Cell Signaling Technology, Danvers, MA, USA); and Bcl-2-associated X (Bax), 2772S (1:500, Cell Signaling Technology).

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Control, Quantitative RT-PCR

Journal: European Journal of Histochemistry : EJH

Article Title: Can the AGE/RAGE/ERK signalling pathway and the epithelial-to-mesenchymal transition interact in the pathogenesis of chronic rhinosinusitis with nasal polyps?

doi: 10.4081/ejh.2020.3079

Figure Lengend Snippet: Immunohistochemistry and semi-quantitative analysis for TGF-β1, Smad2/3, Collagen I-III and α-SMA. No significative differences in immunopositivity between CTRL (A,D,G,L) and CRSwNP (B,E,H,M) samples were found as confirmed by semi-quantitative evaluation (C,F,I,N). Original magnification 10×, scale bar: 100 µm.

Article Snippet: Specimens were incubated overnight at 4°C with the following antibodies: AGE (ab23722: Abcam, Cambridge, UK; dilution 1:500); RAGE (pA1-075: Thermo Fisher Scientific Inc., Waltham, MA, USA; dilution 1:100); p-ERK (sc-7383; Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:200); MMP-3 (sc-6839; Santa Cruz Biotechnology; dilution 1:50); TGF-β1 (sc- 8784; Santa Cruz Biotechnology; dilution 1:200); Smad2/3 (sc- 6202; Santa Cruz Biotechnology; dilution 1:300); Collagen I (sc- 8784; Santa Cruz Biotechnology; dilution 1:200); Collagen III (sc- 8781; Santa Cruz Biotechnology; dilution 1:200); α- SMA (sc- 32251: Santa Cruz Biotechnology; dilution 1:200; E-cadherin (sc- 7870: Santa Cruz Biotechnology; dilution 1:100); Vimentin (sc- 6260: Santa Cruz Biotechnology; dilution 1:100); IL-6 (sc-28343: Santa Cruz Biotechnology; dilution 1:100).

Techniques: Immunohistochemistry