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: Acta Neuropathologica Communications

Article Title: Brain injury drives optic glioma formation through neuron-glia signaling

doi: 10.1186/s40478-024-01735-w

Figure Lengend Snippet: a Following optic nerve crush (ON-CR) at 6 weeks of age, Nf1 flox/flox ; hGFAP-Cre mice at 12 weeks of age have increased numbers of CD3 + cells in their optic nerves relative to those receiving a sham operation ( n = 6); however, there was no change in b Ccl4 mRNA expression by qPCR ( n = 3). c Immediately after ON-CR, Nf1 flox/flox ; hGFAP-Cre mice received intraperitoneal injections of anti-CD3 (αCD3) antibodies every other day for 6 weeks. Control mice were injected with anti-IgG antibodies. Optic nerves were analyzed at 12 weeks of age. αCD3 antibody treatment (150 µg) reduced d CD3 + cell content ( n = 5) and e proliferation (%Ki67 + cells; n = 5). f αCD3 antibody treatment following ON-CR does not change Ccl5 RNA expression by qPCR compared to IgG-treated controls (150 µg; n = 3). Increased Ccl5 RNA expression in the optic nerves (qPCR) was observed 7 days after ON-CR in g Nf1 flox/flox ; hGFAP-Cre ( n = 3), h athymic ( Foxn1 nu/nu ; n = 4), and i Rag1 −/− mice ( n = 4) compared to sham controls. Scale bars: a , d , e 50 µm. Two-tailed Student’s t test (ns, not significant)

Article Snippet: Four-week-old Nf1 OPG mice were treated with 10 mg/kg NFκB inhibitor (NFκB-IN; Caffeic acid phenethyl ester, Fisher Scientific, 274310), 275 mg/kg PLX3397-containing or control chow pellets (Free Base), 1 mg/ml anti-IL-1β neutralizing antibodies (R&D System 1060-DE-100), anti-IgG2a control antibodies (R&D Systems), or memantine hydrochloride (20 mg/kg) by intraperitoneal injection.

Techniques: Expressing, Control, Injection, RNA Expression, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Brain injury drives optic glioma formation through neuron-glia signaling

doi: 10.1186/s40478-024-01735-w

Figure Lengend Snippet: a Top 3 identified pathways enriched in the optic nerves of Nf1 flox/flox ; hGFAP-Cre mice after optic nerve crush (ON-CR) relative to sham controls following filtering of differentially expressed transcripts from bulk RNA sequencing. b Increased IL-1β RNA expression (qPCR) is observed in the optic nerves of 12-week-old Nf1 flox/flox ; hGFAP-Cre mice following ON-CR at 6 weeks of age relative to sham controls ( n = 4). c Representative IL-1β immunostaining in the optic nerves of Nf1 flox/flox ; hGFAP-Cre mice following ON-CR relative to sham control mice. d RNAscope (in situ RNA hybridization) demonstrates that oligodendrocytes (Olig2 + cells) express IL-1β. e Immunostaining reveals that the majority of the Olig2 + cells co-label with CC1, but not with NG2. f Increasing IL-1β concentrations (0–75 ng/ml) increases microglia Ccl5 protein expression in vitro. g IL-1β-induced microglial Ccl5 production is attenuated by 10 mg/kg NFκB inhibitor (NFκB-IN; Caffeic acid phenethyl ester) treatment in vitro ( n = 4). h Anti-IL1β neutralizing antibody treatment (1 mg/ml) of Nf1 flox/flox ; hGFAP-Cre mice immediately after ON-CR at 6 weeks of age results in decreased i optic nerve proliferation (%Ki67 + cells; n = 5) and j Ccl5 mRNA expression (qPCR; n = 3) when analyzed at 12 weeks of age compared to IgG-treated (1 mg/ml) controls. Data are presented as the means ± SEM. Scale bars: c , d , e , i 50 µm. b , c , e , i , j . Two-tailed Student’s t test; f , g One-way ANOVA with Bonferroni post-test correction

Article Snippet: Four-week-old Nf1 OPG mice were treated with 10 mg/kg NFκB inhibitor (NFκB-IN; Caffeic acid phenethyl ester, Fisher Scientific, 274310), 275 mg/kg PLX3397-containing or control chow pellets (Free Base), 1 mg/ml anti-IL-1β neutralizing antibodies (R&D System 1060-DE-100), anti-IgG2a control antibodies (R&D Systems), or memantine hydrochloride (20 mg/kg) by intraperitoneal injection.

Techniques: RNA Sequencing, RNA Expression, Immunostaining, Control, RNAscope, In Situ, Hybridization, Expressing, In Vitro, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Brain injury drives optic glioma formation through neuron-glia signaling

doi: 10.1186/s40478-024-01735-w

Figure Lengend Snippet: a Traumatic brain injury (TBI) in Nf1 flox/flox ; hGFAP-Cre mice at 6 weeks of age results in increased b optic nerve volume ( n = 6) and c proliferation (%Ki67 + cells; n = 7) relative to sham treated mice when analyzed at 12 weeks of age, as well as increased TAMs (%Iba1 + cells) and CD3 + T cell content. Increased d Ccl5 RNA expression (qPCR) and e glutamate levels are observed in the optic nerves of Nf1 flox/flox ; hGFAP-Cre mice ( n = 4) 7 days after TBI compared to sham controls. f αIL-1β neutralizing antibody (1 mg/ml) and h memantine treatment (20 mg/kg) both decrease proliferation (%Ki67 + cells; n = 5) and g , i Ccl5 expression ( n = 3) following TBI relative to their respective control mice (IgG and vehicle treatment groups). Data are presented as the means ± SEM. Scale bar: b 100 μm; c , f , i 50 μm. Two-tailed Student’s t test

Article Snippet: Four-week-old Nf1 OPG mice were treated with 10 mg/kg NFκB inhibitor (NFκB-IN; Caffeic acid phenethyl ester, Fisher Scientific, 274310), 275 mg/kg PLX3397-containing or control chow pellets (Free Base), 1 mg/ml anti-IL-1β neutralizing antibodies (R&D System 1060-DE-100), anti-IgG2a control antibodies (R&D Systems), or memantine hydrochloride (20 mg/kg) by intraperitoneal injection.

Techniques: RNA Expression, Expressing, Control, Two Tailed Test

Journal: Genes

Article Title: Association of Androgenic Regulation and MicroRNAs in Acinar Adenocarcinoma of Prostate

doi: 10.3390/genes13040622

Figure Lengend Snippet: Correlation of the relative prostatic levels of the miRs 27a-3p, 124, 130a, 488-3p, and 506 between the CaP and NPH groups.

Article Snippet: The obtained complementary DNA (cDNA) was stored at −20 °C and later added to 96-well plates with 7.5 μL of 1x TaqMan Universal PCR Master Mix II (Life Technologies, Carlsbad, CA, USA) and 0.5 μL of TaqMan ® Advanced miRNA Assays for hsa-miR-27a-3p (477998_mir ASSAY ID), hsa-miRNA-124 (002197 ASSAY ID), hsa-miRNA-130a (462691_mat ASSAY ID), hsa-miR-488-3p (002357 ASSAY ID), and hsa-miR-506 (001050 ASSAY ID); (TaqMan MGB probe, and forward and reverse primers from Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s recommendations.

Techniques:

Journal: Genes

Article Title: Association of Androgenic Regulation and MicroRNAs in Acinar Adenocarcinoma of Prostate

doi: 10.3390/genes13040622

Figure Lengend Snippet: ROC curves for the miR-27a-3p, miR-124, miR-130a, miR-488-3p, and miR-506.

Article Snippet: The obtained complementary DNA (cDNA) was stored at −20 °C and later added to 96-well plates with 7.5 μL of 1x TaqMan Universal PCR Master Mix II (Life Technologies, Carlsbad, CA, USA) and 0.5 μL of TaqMan ® Advanced miRNA Assays for hsa-miR-27a-3p (477998_mir ASSAY ID), hsa-miRNA-124 (002197 ASSAY ID), hsa-miRNA-130a (462691_mat ASSAY ID), hsa-miR-488-3p (002357 ASSAY ID), and hsa-miR-506 (001050 ASSAY ID); (TaqMan MGB probe, and forward and reverse primers from Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s recommendations.

Techniques:

Journal: Genes

Article Title: Association of Androgenic Regulation and MicroRNAs in Acinar Adenocarcinoma of Prostate

doi: 10.3390/genes13040622

Figure Lengend Snippet: Comparison by the D’Amico score and the relative prostate levels of AR and miRs investigated.

Article Snippet: The obtained complementary DNA (cDNA) was stored at −20 °C and later added to 96-well plates with 7.5 μL of 1x TaqMan Universal PCR Master Mix II (Life Technologies, Carlsbad, CA, USA) and 0.5 μL of TaqMan ® Advanced miRNA Assays for hsa-miR-27a-3p (477998_mir ASSAY ID), hsa-miRNA-124 (002197 ASSAY ID), hsa-miRNA-130a (462691_mat ASSAY ID), hsa-miR-488-3p (002357 ASSAY ID), and hsa-miR-506 (001050 ASSAY ID); (TaqMan MGB probe, and forward and reverse primers from Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s recommendations.

Techniques: Comparison

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Wisp1 activated Akt/MAPK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis maintained for 30 minutes before cells were lysed for protein extraction while cells were treated for 3 hours prior to RNA extraction for comparing EMT marker gene expression. A, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in B16F10 cells. Cells were treated with specific phospho-Akt inhibitor MK-2206 and/or phospho-MAPK inhibitor U0126. Immunoblot for phospho-Akt and phospho-Erk1/2 inhibition was shown on the right upper corner. Pan-Akt and total Erk1/2 were also probed as loading control. B, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in YUMM1.7 cells, with DMSO as control. C, Immunoblot for phospho-Akt and phospho-Erk1/2 in indicated mouse melanoma cells with treatment of recombinant mouse Wisp1 protein (rmWisp1, final 5μg/ml). Cells grown on 6-well plates in complete DMEM for 48 hours and serum-free DMEM (SFM) for another 48 hours before rmWISP1 was added. Pan-Akt and total Erk1/2 were probed as loading control. D, Immunoblot analysis of Akt/MAPK activation in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were grown on 6-well plates in complete DMEM for 48 hours (0 hour point for SFM) and switched to SFM for 24 hour or 48 hours. Indicated cells were treated with rmWisp1 for 30 minutes following 0, 24, or 48 hours in SFM before analyzed for kinase activation. The first lane loaded with YUMM 1.7 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. E, Immunoblot for Akt/MAPK activation in YUMM1.7 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D). The first lane loaded with B16F10 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. F Snai1 activation and E-cadherin repression in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D) except that rmWisp1 treatment at each point maintained for 3 hours. G-H, EMT marker gene expression after Akt/MAPK activation in B16F10-KO1 (G) or YUMM1.7-KO1 (H) by rmWisp1 was blocked by Akt/MAPK inhibitors. rmWisp1 with DMSO or inhibitors was added after indicated cells were grown on 6-well plates in complete DMEM for 48 hours and in SFM for 24 hours. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: The other rabbit polyclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA): anti-β-actin (13E5), anti-Snail (C15D3), anti-ZEB1 (D80D3), anti-N-Cadherin (D4R1H), anti-Phospho-Akt (Ser473) (D9E), anti-Akt (pan) (C67E7), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) and anti-p44/42 MAPK (Erk1/2) (137F5).

Techniques: Marker, Expressing, Western Blot, Protein Extraction, RNA Extraction, Inhibition, Recombinant, Activation Assay, Knock-Out