Journal: Frontiers in Genetics

Article Title: Elevated Serum FGG Levels Prognosticate and Promote the Disease Progression in Prostate Cancer

doi: 10.3389/fgene.2021.651647

Figure Lengend Snippet: FGG knockdown inhibits PCa cell proliferation in vivo . (A) The efficiency of the shRNA. (B) Bioluminescence images of the resultant DU145 tumors in the NC and FGG-KD groups. (C) Subcutaneous tumors were excised and weighed 4 weeks after inoculation. (D) Relative tumor volumes of the NC and FGG-KD groups. (E) Representative IHC and HE staining images of the NC and FGG-KD subcutaneous tumors. * p < 0.05, ** p < 0.01.

Article Snippet: Cells were seeded in six-well plates and were incubated with antibiotic-free medium half an hour before transfection. si-FGG, si-STAT3, and non-targeting control siRNA (si-control) (Synbio Technologies, China) were transfected in Opti-MEM medium (Invitrogen) using X-tremeGENE TM siRNA Transfection Reagent (Roche, Germany) for 6 h according to the manufacturer’s instructions.

Techniques: Knockdown, In Vivo, shRNA, Staining

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