Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MicroRNA-139 inhibits hepatocellular carcinoma cell growth through down-regulating karyopherin alpha 2

doi: 10.1186/s13046-019-1175-2

Figure Lengend Snippet: MiR-139 suppresses HCC migration and invasion. a - e Hep3B and SMMC7721 cells were transfected with 100 nM miR-139 mimic oligos or control oligos. The migration capacity of ( a ) Hep3B and ( b ) SMMC7721 cells were analyzed with the transwell migration assay. The invasion ability of ( c ) Hep3B and ( d ) SMMC7721 cells were measured with the matrigel transwell invasion assay. e The protein expression levels of E-cadherin, Vimentin and SNAIL1 were assayed with western blot in both Hep3B and SMMC7721 cells. f Hep3B and SMMC7721 cells were transfected with 100 nM miR-139 inhibitory or control oligos. The protein levels of E-cadherin, Vimentin and SNAIL1 were measured by western blot in both cell lines. All experiments were repeated 3 times. ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used in this study included: E-cadherin (1:1000, Abcam, USA), Vimentin (1:1000, Boster, China), SNAIL1 (1:1000, Boster, China), KPNA2 (1:1000, Abcam, USA), c-myc (1:1000, Beyotime, China), POU5F1 (1:1000, Abcam, USA), and β-actin (1:2000, Abcam, USA).

Techniques: Migration, Transfection, Control, Transwell Migration Assay, Transwell Invasion Assay, Expressing, Western Blot

Journal: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: HCMV regulates the VDR repressors Snail1 and Snail2. ( A ) RT-qPCR analysis of VDR, Snail1 and Snail2 mRNA expression in mock- and CMV-infected HFF is shown. Asterisks indicate significant differences to the mock control sample (unpaired Student’s t test). ( B ) Immunoblots showing VDR, Snail1, Snail2 and IE1/2 expression compared to GAPDH loading control in HFF infected with CMV and harvested at the indicated time point p.i. Representative blot and quantification from multiple experiments with significant differences to the mock (0 h) control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc tests.

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

Techniques: Quantitative RT-PCR, Expressing, Infection, Western Blot

Journal: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: VDR holoenzyme downregulation is dependent on immediate early gene transcription. ( A ) Vitamin-D-receptor expression was measured in mock- or HCMV-infected HFF at the indicated time points, showing fold change (2 − Δ Δ Ct) compared to mock-infected cells (mean ± SEM). Asterisks indicate significant differences to the mock control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. ( B ) VDR, IE1 (pp72) and GAPDH protein expression were determined by Western blot in HFF that were infected with mock (M), CMV (C) and UV-inactivated CMV (U) inocula and harvested at the indicated times post infection. ( C ) VDR and HCMV immediate early protein expression was determined in lysates of HFF transfected with a non-targeting control siRNA or IE1/2 specific siRNA harvested at 24 h p.i. Representative blot (left) and quantification of bands from multiple experiments relative to the GAPDH loading control (right). Asterisks indicate significant differences to the CMV siCTRL sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. ( D ) HEK293 cells were transfected with pcDNA empty vector control or pcDNA containing the IE2 open reading frame. Lysates were analyzed by Western blot at 48 h post transfection. Asterisks indicate significant differences to empty vector control (unpaired t-test). ( E ) VDR and Snail1 expression was determined at 96 h p.i. in lysates of HFF treated with vehicle (DMSO) or 10 μg/mL Ganciclovir (GCV). HCMV immediate early and pp150 late antigens were stained as a control. ( F ) RXR α protein expression in HFF was determined by specific immunoblots at the indicated time points p.i. Representative blot (left) and quantification of bands from multiple experiments relative to the GAPDH loading control (right, one-way ANOVA and Dunnett’s post-hoc test). ( G ) ARPE-19 cells were infected with mock supernatant or the pentamer-positive (TS15-rN) variant of the Towne strain and harvested 14 d p.i. VDR, Snail1, pp150 late antigen and a GAPDH loading control detection by Western blot is shown.

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

Techniques: Expressing, Infection, Western Blot, Transfection, Plasmid Preparation, Staining, Variant Assay

Journal: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: Snail phosphorylation and regulation by DNA-Protein kinase. ( A ) Lysates from mock- and HCMV-infected cells were harvested at 24 h p.i. and subjected to Phos-Tag SDS-PAGE. As additional controls, CMV-infected HFF were treated with a phosphatase inhibitor for 2 h before lysis (calyculin A), or with lambda phosphatase for 30 min λ ). A representative Snail1-specific immunoblot shows the ratio between unphosphorylated (black arrows) and phosphorylated Snail1 (red circles). The same blot is shown with short (left) and long exposure (right). Height of phosphorylated bands also present in the CMV condition is marked with full red circles, open red circles indicate further (weak) phosphorylation bands in the calyculin A treatment control. ( B ) Representative Western blot showing DNA-PK and Snail protein expression determined in lysates of HCMV-infected HFF transfected with a non-targeting control siRNA or DNA-PK specific siRNAs and harvested at 24 h p.i. (representative Western blot on the left and quantification relative Snail1 band intensity of multiple independent experiments on the right; asterisks show significant differences to the CMV siCTRL sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test).

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

Techniques: Infection, SDS Page, Lysis, Western Blot, Expressing, Transfection

Journal: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: Snail ablation restores VDR expression. ( A ) VDR and Snail protein expression was determined in lysates of HFF transfected with a non-targeting control siRNA or Snail1 specific siRNA and harvested at 24 h p.i. Representative blot (left) and quantification of bands from multiple experiments relative to the GAPDH loading control (right). Asterisks show significant differences to the CMV siCTRL control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. ( B ) Plaque assay showing the percentage of plaques in siRNA-transfected cells compared to an untransfected control. Asterisks show significant differences in two different Snail-targeting siRNAs to the CMV siCTRL control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. Light microscope pictures of representative areas are shown on the right.

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

Techniques: Expressing, Transfection, Plaque Assay, Light Microscopy

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: Snail1, Snail2, and E47 mRNA levels are transiently increased during EGF- or HGF-induced branching morphogenesis. (A) Clusters of mammary epithelial cells were embedded in collagen gel and treated with no growth factor (No GF), EGF, or HGF and monitored for branching at 3, 6, 9, and 24 h. Shown are nuclei (blue) and actin (green). Scale bars, 100 μm. (B) Quantification of the percentage of branching from 20 clusters for No GF, EGF, or HGF treatment; shown are mean±s.e.m. (n=3 independent experiments). **P<0.01 versus No GF (two-way ANOVA with Bonferroni comparison). (C–F) Total RNA was isolated at indicated times and used to determine the mRNA levels of (C) Snail1, (D) Snail2, (E) E47, and (F) E-cadherin by qRT–PCR. The mRNA levels were normalized to the levels of β-actin in each sample and each value was expressed relative to the levels in No GF; shown are mean±s.e.m. (n=3). *P<0.05; **P<0.01 versus 3 h No GF (two-way ANOVA with Bonferroni comparison).

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Comparison, Isolation, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: Snail1, Snail2, and E47 are increased at branch sites. (A) Microfabricated mammary epithelial tubules were treated with no growth factor (No GF), EGF, or HGF for 24 h. Phase contrast images and frequency maps of 60 tubules stained for nuclei are shown. Scale bars, 50 μm. Colour bars indicate frequency. (B) Branching was quantified by measuring the pixel intensity 12 μm away from the tip of tubules; shown are mean±s.e.m. (n=5). **P<0.01 versus No GF (one-way ANOVA with Bonferroni comparison). (C) Microfabricated mammary epithelial tubules were treated with No GF or EGF for 8 h and stained for Snail1, Snail2, or E47. Scale bars, 50 μm. (D) Frequency maps of 50 tubules stained for Snail1, Snail2, E47, or vimentin. Scale bars, 50 μm. Colour bars indicate frequency. (E) Microfabricated mammary epithelial tubules were treated with No GF or EGF for 8 or 24 h and stained for E-cadherin. Scale bars, 50 μm.

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Staining, Comparison

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: Loss of Snail1, Snail2, or E47 represses branching morphogenesis. (A–F) Mammary epithelial cells were transfected with two independent shSnail1, three independent shSnail2, three independent shE47, scrambled shRNA (Sc), or no shRNA cassette (NT). After 48 h, transfected cells were treated with no growth factor (No GF), EGF, or HGF for 9 h. Total RNA was isolated for determination of (A) Snail1, (B) Snail2, (C) E47, or (D–F) E-cadherin mRNA levels by qRT–PCR. The mRNA levels were normalized to the levels of β-actin in each sample and each value was expressed relative to the levels in No GF of scrambled shRNA transfected cells (Sc); plotted are mean±s.e.m. (n=3). *P<0.05; **P<0.01 versus Sc (No GF) (two-way ANOVA with Bonferroni comparison). (G) Mammary epithelial cells were transfected with shSnail1, shSnail2, shE47, Sc, or NT and used to generate microfabricated mammary epithelial tubules. Tubules were treated with No GF, EGF, or HGF for 24 h. Frequency maps of branching from 50 tubules are shown. Scale bars, 50 μm. Colour bar indicates frequency. (H) Branching was quantified as described above; shown are mean±s.e.m. (n=5). All results were confirmed with at least two independent shRNA constructs for each gene. **P<0.01 versus NT (No GF); ##P<0.01 versus Sc (EGF) (two-way ANOVA with Bonferroni comparison).

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Transfection, shRNA, Isolation, Quantitative RT-PCR, Comparison, Construct

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: Snail1, Snail2, and E47 induce mammary epithelial branching morphogenesis. (A–C) Mammary epithelial cells were transfected with Flag-tagged Snail1, Snail2, E47, YFP, or nothing (NT). (A) Total protein was assayed by immunoblot to determine the expression levels of Snail1, Snail2, E47, or E-cadherin. Total RNA was isolated for determination of (B) E-cadherin or (C) N-cadherin mRNA levels by qRT–PCR. The mRNA levels were normalized to the levels of β-actin in each sample and each value was expressed relative to the levels in Flag-tagged YFP transfected cells; shown are mean±s.e.m. (n=4). *P<0.05; **P<0.01 versus Flag-tagged YFP (one-way ANOVA with Bonferroni comparison). (D) Mammary epithelial cells were transfected with Flag-tagged Snail1, Snail2, E47, YFP, or NT and used to generate microfabricated mammary epithelial tubules. Frequency maps of branching from 50 tubules are shown. Scale bars, 50 μm. Colour bar indicates frequency. (E) Branching was quantified as described above; shown are mean±s.e.m. (n=5). **P<0.01 versus NT (No GF) (two-way ANOVA with Bonferroni comparison).

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Transfection, Western Blot, Expressing, Isolation, Quantitative RT-PCR, Comparison

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: Loss of Snail1 or Snail2 leads to apoptotic cell death at branch sites during mammary epithelial branching morphogenesis. (A, B) Mammary epithelial cells were transfected with shSnail1, shSnail2, shE47, or scrambled shRNA (Sc). After 48 h, transfected cells were treated with no growth factor (No GF), EGF, or HGF for 9 h. Total RNA was isolated for determination of (A) p53 and (B) BID mRNA levels using qRT–PCR. The mRNA levels were normalized to the levels of β-actin in each sample and each value was expressed relative to the levels in No GF of scrambled shRNA transfected cells; shown are mean±s.e.m. (n=3). **P<0.01 versus SC (No GF) (two-way ANOVA with Bonferroni comparison). (C) Mammary epithelial cells were transfected with shSnail1, shSnail2, shE47, or scrambled shRNA (Sc) and used to generate microfabricated mammary epithelial tubules. Tubules were treated with No GF, EGF, or HGF for 20 h and fixed and stained for cleaved caspase-3 and nuclei. Scale bars, 50 μm. (D) Percent area of active caspase-3 was quantified by measuring cleaved caspase-3-positive areas in 25 tubules from at least three independent experiments; shown are mean±s.e.m. **P<0.01 versus NT (No GF) (two-way ANOVA with Bonferroni comparison). (E) Mammary epithelial cells were transfected with shSnail1, shSnail2, scrambled RNA (Sc), or nothing (NT). After 24 h, cells were treated with or without EGF. Total protein was analysed by immunoblot to determine the expression levels of p53 or β-actin. (F) Microfabricated mammary epithelial tubules were generated and treated with or without EGF and stained for p53. Scale bars, 50 μm. (G) Microfabricated mammary epithelial tubules were generated and treated with EGF for the indicated time. Percent area of cleaved caspase-3 in scrambled RNA (Sc), shSnail1, or shSnail2-transfected samples was quantified as described above; shown are mean±s.e.m. (n=30). *P<0.05; **P<0.01 versus Sc (0 h) (two-way ANOVA with Bonferroni comparison).

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Transfection, shRNA, Isolation, Quantitative RT-PCR, Comparison, Staining, Western Blot, Expressing, Generated

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: Inhibition of apoptosis by Snail1 or Snail2 is not sufficient to induce mammary branching morphogenesis. (A, B) Mammary epithelial cells were transfected with Flag-tagged Snail1, Snail2, E47, YFP, or nothing (NT). Total RNA was isolated for determination of (A) p53 and (B) BID mRNA levels using qRT–PCR. The mRNA levels were normalized to the levels of β-actin in each sample and each value was expressed relative to the levels in Flag-tagged YFP; shown are mean±s.e.m. (n=4). *P<0.05; **P<0.01 versus Flag–YFP (one-way ANOVA with Bonferroni comparison). (C) Mammary epithelial cells were transfected with Flag-tagged Snail1, Snail2, or YFP and used to generate microfabricated mammary epithelial tubules. Tubules were cultured for 20 h and stained for cleaved caspase-3 and nuclei. Scale bars, 50 μm. (D) Percent area of cleaved caspase-3 was quantified as described above; shown are mean±s.e.m. (n=30). **P<0.01 versus Flag-tagged YFP (one-way ANOVA with Bonferroni comparison). (E, F) Microfabricated mammary epithelial tubules were treated with caspase-3 inhibitor (Z-DEVD-FMK, 20 μM) or vehicle (DMSO). (E) Tubules stained for cleaved caspase-3 (red) and nuclei (blue) and (F) frequency maps of 50 tubules are shown. Scale bars, 50 μm. (G) Microfabricated mammary epithelial tubules generated from cells transfected with shSnail1-a or shSnail2-d were treated with caspase-3 inhibitor (Z-DEVD-FMK, 20 μM) or vehicle. Frequency maps of branching from 50 tubules are shown. Scale bars, 50 μm. Colour bar indicates frequency.

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Inhibition, Transfection, Isolation, Quantitative RT-PCR, Comparison, Cell Culture, Staining, Generated

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: Repressing E-cadherin expression is required for branching morphogenesis. (A) Mammary epithelial cells were transfected with E-cadherin or empty vector (EV) and incubated with EGF or HGF for 24 h. Total protein was assayed by immunoblot to determine the expression levels of E-cadherin. (B) Relative intensity of bands in immunoblots shown in panel (A). **P<0.01 versus EV of each treatment (Student's t-test). (C) Mammary epithelial cells were transfected with E-cadherin or EV and used to generate microfabricated mammary epithelial tubules. Frequency maps of branching from 50 tubules are shown. Scale bars, 50 μm. Colour bar indicates frequency. (D) Branching was quantified as described above; shown are mean±s.e.m. (n=4). **P<0.01 versus EV (No GF); ##P<0.01 versus EV (EGF) (two-way ANOVA with Bonferroni comparison). (E) Mammary epithelial cells were co-transfected with Flag-tagged Snail1 and E-cadherin or EV. Total protein was assayed by immunoblot to determine the expression levels of Snail1 or E-cadherin. (F) Mammary epithelial cells were co-transfected with Flag-tagged Snail1 and E-cadherin or EV and used to generate microfabricated mammary epithelial tubules. Frequency maps of branching from 50 tubules are shown. Scale bars, 50 μm. Colour bar indicates frequency. (G) Branching was quantified as described above; shown are mean±s.e.m. (n=4). **P<0.01 versus NT (one-way ANOVA with Bonferroni comparison). (H) Mammary epithelial cells were co-transfected with Flag-tagged E47 and E-cadherin or EV. Total protein was assayed by immunoblot to determine the expression levels of E47 or E-cadherin. (I) Mammary epithelial cells co-transfected with Flag-tagged E47 and E-cadherin or EV were used to generate microfabricated mammary epithelial tubules. Frequency maps of branching from 50 tubules are shown. Scale bars, 50 μm. Colour bar indicates frequency. (J) Branching was quantified as described above; shown are mean±s.e.m. (n=4). **P<0.01 versus NT (one-way ANOVA with Bonferroni comparison).

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Comparison

Journal: The EMBO Journal

Article Title: Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis

doi: 10.1038/emboj.2011.159

Figure Lengend Snippet: The role of Snail1, Snail2, and E47 in mammary epithelial branching morphogenesis. Mammary epithelial tissues are quiescent in the absence of exogenous stimuli. In response to growth factor stimulation, mammary epithelial cells located at branch sites express Snail1, Snail2, and E47 to induce expression of mesenchymal markers and promote cell survival. Consequently mammary epithelial cells initiate branching.

Article Snippet: These transcription factors are involved in multiple processes during development and mice deficient for Snail1 or Twist1 die during embryogenesis ( Chen and Behringer, 1995 ; Carver et al., 2001 ).

Techniques: Expressing