Journal: Journal of Virology

Article Title: Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

doi: 10.1128/JVI.00970-14

Figure Lengend Snippet: (A) Schematic of the Jc1 (genotype 2a) virus and luciferase reporter replicons (Luc-JFH1 [genotype 2a] and Luc-Con1 [genotype 1b]) used to determine the role of the glycosphingolipid machinery in HCV replication. Note that NS5A has a C-terminal mCherry fusion (in Luc-JFH1 and virus used for panel B) as reported by Gottwein et al. (36). (B) Huh7.5 cells were mock infected or infected with HCV J6/JFH1 (MOI of 0.1) with a C-terminal mCherry fusion to NS5A as described for panel A (36). At 48 h postinfection, the cells were processed for confocal microscopy with mouse monoclonal αPI4P antibody (green). NS5A was detected via mCherry fluorescence. Alternatively, HCV Con1 replicon cells were grown for 48 h and stained with mouse monoclonal α-PI4P antibody (red) and rabbit polyclonal antibody against NS4B (green). The boxed areas are a magnified view for colocalization (yellow) of HCV NS5A or NS4B protein with PI4P. (C) Diagram of the de novo biosynthetic pathway leading to sphingolipids (e.g., ceramide and sphingomyelin) and glycosphingolipids (e.g., glucosylceramide and lactosylceramide) production. The SPTLC1, 2, 3 complex encodes the subunit of SPT (highlighted in gray), the first enzyme in the pathway leading to ceramide production. The SPTLC1 subunit interacts with SPTLC2 or SPTLC3 to form two distinct enzymatic functional complexes. Notice that ceramide is an intermediate product for generating both sphingolipids and glycosphingolipids. UGCG is highlighted in gray and codes for glucosylceramide synthase, a rate-limiting enzyme in glycosphingolipid synthesis. NB-DNJ and PDMP (54,–59) are two pharmacological inhibitors of UGCGC. FAPP2 is highlighted in gray and carries glucosylceramide from the cis-Golgi to the trans-Golgi network for conversion into lactosylceramide and other glycosphingolipids. CoA, coenzyme A.

Article Snippet: Rabbit polyclonal antibody specific for FAPP2 was obtained from Abcam Inc., (Cambridge MA).

Techniques: Luciferase, Infection, Confocal Microscopy, Fluorescence, Staining, Functional Assay

Journal: Journal of Virology

Article Title: Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

doi: 10.1128/JVI.00970-14

Figure Lengend Snippet: Lactosylceramide is associated with HCV NS4B protein. (A) Parental Huh7.5 and HCV Con1 (genotype 1b) replicon cells were grown for 48 h and processed for confocal microscopy with mouse monoclonal αLacCer antibody (1:500; green) and rabbit polyclonal αNS4B antibody (1:150; red). Huh7.5 cells also were infected with HCV Jc1 (MOI of 1) and processed for confocal microscopy as described above. The boxed areas represent a magnified view for colocalization (yellow) of lactosylceramide and HCV NS4B protein. (B) Huh7.5 cells were infected with HCV Jc1 (MOI of 1) as described for panel A and processed for confocal microscopy 24 h, 48 h, and 72 h postinfection. For each infection time point, confocal images were taken of 20 representative cells. The intensity of lactosylceramide pixels was calculated with the JACoP plugin in ImageJ software. Each filled circle or square (24 h; mock or Jc1 infected), upper or lower triangle (48 h; mock or Jc1 infected), diamond (72 h; mock), or open circle (72 h; Jc1 infected) represents one cell.

Article Snippet: Rabbit polyclonal antibody specific for FAPP2 was obtained from Abcam Inc., (Cambridge MA).

Techniques: Confocal Microscopy, Infection, Software

Journal: Journal of Virology

Article Title: Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

doi: 10.1128/JVI.00970-14

Figure Lengend Snippet: FAPP2 colocalizes with HCV NS5A and viral dsRNA. (A) Parental Huh7.5 and HCV Con1 (genotype 1b) replicon cells were grown for 48 h and processed for confocal microscopy with mouse monoclonal αNS5A antibody (1:1,000; red) and rabbit polyclonal αFAPP2 antibody (1:100; green). (B) Huh7.5, Con1 replicon, or Jc1 (34) virus-infected cells were grown as described for panel A and processed for confocal microscopy with mouse monoclonal antibody against dsRNA (1: 200; red) and rabbit polyclonal αFAPP2 antibody (1:100; green). The boxed areas indicate the magnified view for colocalization (yellow color) of HCV NS5A (A) or dsRNA (B) with FAPP2 protein. (C) FAPP2 cofractionates with HCV replicase NS5A protein in the detergent-resistant membrane fraction. Con1 replicon cell lysates were left untreated or were treated with 1% NP-40 on ice and subjected to membrane floatation. Proteins from pooled fractions (1 to 4 and 5 to 9) were separated by SDS-PAGE, followed by immunoblotting with antibodies against FAPP2, SPTLC1, NS5A, calnexin, or GAPDH. Numbers 1 to 4 refer to membrane (M) fractions, and numbers 5 to 8 refer to soluble (S) fractions.

Article Snippet: Rabbit polyclonal antibody specific for FAPP2 was obtained from Abcam Inc., (Cambridge MA).

Techniques: Confocal Microscopy, Infection, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

doi: 10.1128/JVI.00970-14

Figure Lengend Snippet: FAPP2 knockdown disrupts HCV NS4B and NS5A focus formation. (A) Control and FAPP2 shRNA cells were treated with 3 μg/ml doxycycline as described in the legend to Fig. 7, followed by transfection with pIRES vector expressing HCV NS3-4A-4B-5A-5B polyprotein in the presence of doxycycline. At 48 h posttransfection, the cells were fixed and processed for confocal microscopy with mouse monoclonal αNS5A antibody (1:1,000; green). Red fluorescent protein (RFP) indicates control and FAPP2 shRNA cells. Magnified areas, with NS5A subcellular distribution, are shown by rectangles. (B) Control (i to x) and FAPP2 (xi to xx) shRNA cells were treated as described for panel A and processed for confocal microscopy with mouse monoclonal αNS5A antibody (1:1,000; magenta) and rabbit polyclonal antibody against NS4B (1:25; green). RFP indicates shRNA-expressing cells as described for panel A. Magnified areas, with putative NS4B and NS5A colocalization (white), are indicated by rectangles.

Article Snippet: Rabbit polyclonal antibody specific for FAPP2 was obtained from Abcam Inc., (Cambridge MA).

Techniques: shRNA, Transfection, Plasmid Preparation, Expressing, Confocal Microscopy

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high‐throughput screening. B Upper panel, sequence interaction of miR‐182‐3p with the target site of the wild type 3′UTR of TRF2 in human. Bottom panel, generation of mutant 3′UTR of TRF2 luciferase construct containing the deletion of target site for miR‐182‐3p. C–E Luciferase reporter assay in HeLa cells using the synthetic miR‐Control or miR‐182‐3p in combination with the wild type (C) or the mutant 3′UTR of TRF2 construct (D) or the wild type 3′UTR of TRF1 (E). F, G Western blotting for TRF2 expression in telomerase‐positive (HeLa, HCT116, MDA‐MB‐231, MDA‐MB‐436) and ALT‐positive (U2‐OS, Saos‐2) cells transiently transfected with miR‐Control or miR‐182‐3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. H U2‐OS cells transiently transfected with the miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor were assayed by quantitative immunofluorescence for TRF2 3 days post‐transfection. Left panel, representative images. Scale bar: 10 μm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N = number of analyzed nuclei. Red bar indicates mean value. I U2‐OS cells transfected as described in (H) were assayed by immunofluorescence combined with telomeric FISH. Left panel, representative images of co‐localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10 μm. Right panel, co‐localizations were analyzed using ImageJ software. N = number of analyzed nuclei. Data information: For (C–G and I), data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by Student's t ‐test; for (H), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Luciferase, Software, In Silico, High Throughput Screening Assay, Sequencing, Mutagenesis, Construct, Reporter Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Results of high‐throughput luciferase screening performed in Hela cells using the wild type 3′UTR‐TRF2 vector in combination with each of the 54 miRNAs selected by in silico analysis. Three days post‐transfection, luciferase ratio (Renilla:Firefly) of each miRNA was calculated, the control miRNA was set “1.” Renilla:Firefly ratios < 1 indicate target specificity of candidate miRNAs for the 3′UTR of TRF2. miRNAs near to the ratio of 0.5 were considered for further analysis. Two biological replicates were performed. B HeLa cells transiently transfected with the indicated miRNAs (miR‐Control, miR‐182‐3p, miR‐519e‐5p, miR‐296‐3p) were assayed by western blotting. Upper panel, quantification of TRF2 expression. Bottom panel, representative images of TRF2, TRF1 and RAP1 are shown, actin was used as loading control. C Analysis of TRF2 mRNA expression performed by qPCR in four different cancer cell lines (HeLa, MDA‐MB‐231, MDA‐MB‐436, U2‐OS) 3 days post‐transfection with miR‐Control or miR‐182‐3p. The control miRNA was set “1.” Three independent experiments were performed. D, E Telomeric ChIP assay in MDA‐MB‐231 (D) and U2‐OS cells (E). Quantification of TRF2 enrichment at telomeric repeats, in the different conditions, is shown in the table under the respective figure. Alu probe and Rabbit IgG were used as negative control for the assay. Data information: For (A), data are presented as mean values. For (B, C), data are presented as mean values ± SD and Student t‐ test was used to calculate statistical significance. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: High Throughput Screening Assay, Luciferase, Plasmid Preparation, In Silico, Transfection, Western Blot, Expressing, Negative Control

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A MDA‐MB‐231 cells were transiently transfected with the indicated miRNAs or siRNA. The indicated DNA damage markers were assayed by western blotting. Actin was used as loading control. B Telomeric DNA FISH performed in MDA‐MB‐231 transiently transfected with the indicated miRNAs. Telomere length was measured by TLF software and indicated as arbitrary fluorescence unit (a.f.u). N = number of analyzed nuclei. Black bar indicates mean value. C DNA damage markers were assayed by western blotting in HeLa cells. Actin was used as loading control. D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. Co‐localizations of γH2AX with telomeres are indicated as mean number of TIFs per nucleus. E Representative images and enlargements of co‐localizations of experiment described in D. F Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. G Representative images of co‐localizations relative to the experiment described in (F). H, I MDA‐MB‐231 and HeLa cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with miR‐Control or miR‐182‐3p. TRF2, pATM and γH2AX expression were assayed by western blotting. Actin was used as loading control. Data information: For (D) and (F), data are presented as mean values ± SD. Three independent replicates were performed. Scale bar: 10 μm. At least 60 nuclei were analyzed in (D) and (F). A Student t‐ test was used to calculate statistical significance. For (B), P values are determined by Mann–Whitney t ‐test. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Western Blot, Software, Fluorescence, Immunofluorescence, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analyzed. Representative images and enlargements of co‐localizations (white arrowheads) relative to the experiment described in (A). Scale bar: 10 μm. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images of co‐localizations (white arrowheads) relative to the experiment described in (C). Scale bar: 10 μm. Quantification of TIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. Representative images and enlargements relative to the experiment described in (E). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Quantification of PIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images relative to the experiment described in (G). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Data information: For (A, C, E, G) data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. At least 60 nuclei were analyzed for each experimental condition. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐436 and MDA‐MB‐231 cells underwent two rounds of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24 h up to a maximum of 3 days. The percentage of cell confluence was analyzed. C, D Cell number of MDA‐MB‐436 (C) and MDA‐MB‐231 (D) cells and TRF2 expression were analyzed by automatic cell count and by western blotting at the end of the experiment described in (A) and (B). Actin was used as loading control. E Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐436 at the end of the second cycle of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Red boxes indicate early and late apoptotic cells. F Quantification of Annexin V‐positive cells (%) of experiment described in (E). G Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐231 as described in (E). H Quantification of Annexin V‐positive cells (%) of experiment described in (G). I, J MDA‐MB‐436 cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72 h post‐transfection. Data information: For (A, B) data are shown as mean ± SEM. For (C, D, F, H, I, J), data are shown as mean ± SD. For (A–D) and (I), three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. For (F), (H) and (J), two different biological replicates were performed. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Cell Counting, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Western blotting for TRF2 expression in BJ cells transiently transfected with miR‐Control or miR‐182‐3p. The graph represents the quantification of three independent experiments. Representative images are shown, Actin was used as loading control. Unspecific bands are indicated with (*). B, C Mean of γH2AX foci per nucleus was analyzed in BJ cells 72 h post‐transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in (C). D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72 h post‐transfection with the indicated miRNAs. Left panel: The mean number of TIFs per nucleus was analyzed. Right panel: Representative images and enlargements of co‐localizations. E Cell number of BJ cells was analyzed by automatic cell count at the end of the second round of transfection with miR‐Control or miR‐182‐3p. F FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in (E). G β‐Galactosidase assay in BJ cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. H–J IL‐6 (H), CXCL1 (I), IL‐8 (J) factors were analyzed by ELISA to evaluate the senescence‐associated secretory phenotype (SASP) in BJ cells treated as indicated in (G). Data information: For (A, B, D, E and G–J), a student t‐ test was used to calculate statistical significance. Scale bars (10 μm). P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A TRF2 and γH2AX expression after two rounds of transfection with the indicated miRNAs, was analyzed by western blotting in MCF10A cells. Actin was used as loading control. B–E The mean number of γH2AX foci (B) and TIFs (D) per nucleus were analyzed 72 h post‐transfection with the indicated mimic miRNAs in MCF10A cells. Representative images (C) and (E) are referred to the experiment showed in (B) and (D) respectively. F, G Cell confluence (F) of MCF10A was monitored by Incucyte, every 24 h starting from the day of the second transfection, and cell number (G) was counted at the end of experiment (day 4). H–I Cell cycle progression analysis by PI staining (H) and cell death analysis by Annexin V assay (I) were performed in MCF10A upon two rounds of transfection with the indicated miRNAs. J β‐Galactosidase assay in MCF10A cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. Data information: Panels (B, D, F, G, J) data are presented as mean values ± SD. A Student t‐ test was used to calculate statistical significance. P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Transfection, Western Blot, Staining, Annexin V Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐231 (A) and MDA‐MB‐436 (B) tumor xenografts were treated with LNPs‐empty, LNPs‐miR‐Control or by LNPs‐miR‐182‐3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20 μg of LNPs‐miR‐Control, LNPs‐miR‐182‐3p or equivalent volume of LNPs‐empty as indicated in the scheduling. The mean of tumor volumes ( n = 5 per group) is shown. C, D Tumors from mice treated in (A) and (B) were processed to measure miR‐182‐3p expression by TaqMan qPCR. E Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA‐MB‐231 human breast cancer xenografts. Scale bar: 50 μm. F The histograms show the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, TUNEL or CD31 staining. The analyses were performed on three mice per group, and the points represent the number of field analyzed for each condition. G, H Luminescent MDA‐MB‐436 cells were injected into the brain and monitored by IVIS imaging system. After 1 week from implant, treatment with LNPs‐miR‐Control and LNPs‐miR‐182‐3p was performed as indicated in (A) and (B). Representative images from in vivo (upper panel) or ex‐vivo (bottom panel) brain tumors are shown in (G). Boxplots (H) show the measurement of photons for each brain tumor ( n = 5 per group) acquired at the indicated times. Data information: For (A, B, F), data are shown as mean ± SD. For (C, D, H), the line in the middle of the box plot denotes a median value, the limits of box represent the interquartile range (25 th to 75 th percentiles), while, the whiskers denote the minimum to maximum values. For (A–D) and (H), P values are determined by unpaired two‐tailed t‐ test; for (F), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, TUNEL Assay, Staining, Injection, Imaging, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: The organs (brain, liver, kidney) taken from mice, previously engrafted with MDA‐MB‐231 cells and treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p, were assayed for miR‐182‐3p expression by TaqMan qPCR. Representative images show IHC analysis on tumor samples, from mice bearing MDA‐MB‐436 human breast cancer xenografts, with the indicated markers. Scale bar: 50 μm. The histograms show the expression of TRF2 indicated as immunoreactivity score (IRS) and the percentage of positive cells to γH2AX, TIUNEL or CD31 staining in MDA‐MB‐436 xenografts. Three mice per group were analyzed, the points represent the number of field analyzed for each condition. Data information: For (A, C), data are presented as mean values ± SD. Statistical significance using unpaired (A) or Mann–Whitney t‐ test (C) was calculated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B PDTCs #1 and #2 underwent two rounds of transfection with miR‐Control or miR‐182‐3p. Three days after the second transfection, miR‐182‐3p and TRF2 expression were analyzed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. C, D Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50 μm. At least 85 3D cells were analyzed for each experimental condition. E NSG mice implanted with breast PDTX (#2) were treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p as indicated in the scheduling. Caliper measurement of tumors was taken at the indicated days. The mean of tumor volumes ( n = 5 per group) is shown. F miR‐182‐3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. G Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in (E). Scale bar: 50 μm. H The histograms show the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and TUNEL. The analysis was performed on three mice per group, the points represent the number of field analyzed for each condition. Data information: For (A–F) and (H), data are shown as mean ± SD. For (A–F), P values are determined by unpaired two‐tailed t‐ test; for (H), P values are determined by Mann–Whitney t ‐test. For the experiments showed in (A, B) and (C, D) two or three biological replicates were performed, respectively. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Western Blot, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Representative images of intestine sections from mice previously treated with LNPs‐Empty or LNPs‐miR‐182‐3p. H&E staining (scale bar: 200 μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50 μm). B, C Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX‐positive cells (%) (C) on intestine samples. D Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of skin samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). E, F Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX‐positive cells (%) (F) on skin samples. G Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). H, I Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX‐positive cells (%) (I) on bone marrow samples. Data information: For (B, C, E, F, H, I), data are shown as mean ± SD. A Mann–Whitney test t‐ test was used to calculate statistical significance. Four mice per group were analyzed, the points represent the number of field analyzed for each condition. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

doi: 10.7554/eLife.58020

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-RIF1 , Bethyl Laboratories , Cat#A300-568A RRID: AB_669806 , WB (1:5000).

Techniques: Derivative Assay, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Recombinant, Sequencing, Luciferase, Cloning, Imaging, Antibody Labeling, Software, Microscopy

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Preparation and characterization of the cell lines used in the present study. (A) Detection of SMS1, SMS2, and GAPDH in JAR, JAR4, JEG3, and HeLa-mCAT#8 cell lines by immunoblotting. (B) Nucleotide sequences around the target region for the SGMS1 gene-specific sgRNA in exon 9 of the SGMS1 gene of the SMS1KO22 clone (clone 22) were aligned with those of the parental JAR4 cells (parent). Target sequences for the sgRNA and following protospacer adjacent motifs are indicated by underlined and boxed sequences of the parental JAR4 cells, respectively. The SMS1KO22 clone has a homozygous 7-nucleotide deletion in the SGMS1 gene. (C) Detection of SMS1, SMS2, and GAPDH in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2 by immunoblotting. (D) Detection of clustered SM on the surface of JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Cells were treated with EGFP-NT-lysenin and analyzed by flow cytometry. Histograms with magenta line and gray fill represent EGFP-NT-lysenin-treated and untreated cells, respectively. Cells in the M1 region (fluorescent intensity of 50 and above) were defined as positive for binding to EGFP-NT-lysenin. The means and standard deviations of triplicate samples are reported in panel E. (E) Significant differences as determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (F) Quantification of sphingolipids in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2 by LC-MS analysis. Lipids were extracted from cells and quantified by LC-MS. The graphs indicate the means and standard deviations of triplicate samples. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. CMH, ceramide monohexoside (glucosylceramide and galactosylceramide); CDH, ceramide dihexoside (lactosylceramide and galabiosylceramide); Gb3, trisaccharide globo-series sphingolipid; GM3, monosialodihexosylganglioside.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Binding Assay, Comparison, Liquid Chromatography with Mass Spectroscopy

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of SGMS1 gene knockout on RuV growth. (A) Growth kinetics of RuV in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Supernatants of each cell line inoculated with the RuV TO-336WT strain at an MOI of 10 were harvested at 0, 1, 2, 3, or 4 days after inoculation. Infectious titers in the supernatants are represented as means and standard deviations of triplicate samples. (B) Fluorescent microscopy images of each cell line, NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, or SMS1KO22/SMS2, inoculated with the RuV TO-336WT strain at 3 days after inoculation. Green signals indicate the expression of the p150-AG1 protein. Nuclei were stained by DAPI (blue). (C) The rate of p150-AG1-expressing cells inoculated with the RuV TO-336WT strain under the same conditions as for panel B. Cells detached with trypsin-EDTA and fixed with 4% paraformaldehyde were analyzed by flow cytometry. The graph indicates the means and standard deviations of triplicate samples. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: *, P < 0.05; ****, P < 0.0001; ns, not significant. (D and E) Growth kinetics of SINV (D) and MeV (E) in NT1 or SMS1KO22 cells. Infectious titers of progeny viruses are represented as means and standard deviations of triplicate samples.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Gene Knockout, Derivative Assay, Microscopy, Expressing, Staining, Flow Cytometry, Comparison

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of knockout of the SGMS1 or SGMS2 gene on infectivity and entry of RuV in HeLa cells. (A) Detection of SMS1, SMS2, and GAPDH in HeLa-mCAT#8 cell line (Parent) and its gene-edited clones, SGMS1 or SGMS2 single-knockout (ΔSMS1 or ΔSMS2) and double-knockout (DKO) cells, by immunoblotting. (B) Growth kinetics of RuV in HeLa-derived cell lines. Each cell line was inoculated with the TO-336WT-AG1 RuV strain at an MOI of 10, and the supernatants were harvested at the indicated days after incubation. The infectious titers of RuV in the supernatants are represented as means and standard deviations of triplicate samples. (C) Infectivity of pseudotyped VSV in each cell line. Each cell line was inoculated with firefly luciferase gene-coding pseudotyped VSVs VSVFLuc-ΔG (ΔG), VSVFLuc-RV/CE2E1 (RuV-CE2E1), or VSVFLuc-G (VSV-G). The firefly luciferase activity was measured at 24 h postinoculation. The graph indicates the means and standard deviations of three independent assays. Significant differences as determined by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Knock-Out, Infection, Clone Assay, Double Knockout, Western Blot, Derivative Assay, Incubation, Luciferase, Activity Assay, Comparison

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of SGMS1 gene knockout on genome replication, entry, and binding of RuV. (A) Reporter assay of the RuV-subgenomic replicon. NT1 or SMS1KO22 cell line was transfected with the subgenomic replicon RNA HS-Rep-P2R (SGR) or replication-defective mutant HS-Rep-GND-P2R (GND), which expressed Rluc as a reporter, together with mRNAs encoding the RuV-C protein and firefly luciferase (Fluc). After 72 h of transfection, RLuc activity was determined and normalized by Fluc activity. The graph indicates the means and standard deviations of three independent assays. Significant differences as determined by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (B) Infectivity of pseudotyped VSVs in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Each cell line was inoculated with Fluc gene-encoding pseudotyped VSVs, VSVFLuc-ΔG (ΔG), VSVFLuc-RV/CE2E1 (RuV-CE2E1), or VSVFLuc-G (VSV-G). The Fluc activity was measured at 24 h postinoculation. The graph indicates means and standard deviations of three independent assays. Significant differences by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (C) Binding of RuV to JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Each cell line was incubated with RuV at an MOI of 4 on ice for 1 h and then washed to remove unbound viruses. Total RNA was extracted from the cells, and the amount of RuV genomic RNA was determined by quantitative RT-PCR and normalized by the amount of total RNA. The graph indicates the means and standard deviations of three independent assays. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: **, P < 0.01; ****, P < 0.0001; ns, not significant.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Gene Knockout, Binding Assay, Reporter Assay, Transfection, Mutagenesis, Luciferase, Activity Assay, Comparison, Infection, Derivative Assay, Incubation, Quantitative RT-PCR

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Penetration of the RuV genome into the cytoplasm. NT1 or SMS1KO22 cells (A and B), or SMS1KO22cells, SMS1KO22/SMS1-WT clone 1, or SMS1KO22/SMS2 clone 1 (C and D) were inoculated with RuV. After incubation at 37°C for 3 h, cells were fixed with 4% paraformaldehyde. For the control experiment in panel A, NT1 cells were incubated with a medium containing BAPTA-AM (final concentration, 50 μM) before inoculation of RuV at 1 h. The RuV genome (pseudocolored in magenta) and the E1 protein (green) were stained by in situ hybridization and indirect immunofluorescence assay, respectively. Nuclei were stained by DAPI (blue). In panels A and C, representative z -stack images are shown. The three columns on the right are enlarged images of the areas enclosed by dashed boxes in the left column. Bars for original and enlarged images indicate 20 μm and 5 μm, respectively. In panels B and D, percentages of puncta in which RuV genomic RNA is present and colocalized with E1 protein are indicated. The graphs indicate the means and standard deviations of three independent assays. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: **, P < 0.01; *, P < 0.05.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Incubation, Control, Concentration Assay, Staining, In Situ Hybridization, Immunofluorescence, Comparison

Journal: iScience

Article Title: Long noncoding RNA Lnc-DIF inhibits bone formation by sequestering miR-489-3p

doi: 10.1016/j.isci.2022.103949

Figure Lengend Snippet:

Article Snippet: Ser465/Ser467 phosphorylated SMAD2 Rabbit polyclonal antibody , Bioss , Cat# bs-3419R, RRID: AB_10880886.

Techniques: Luciferase, Plasmid Preparation, Recombinant, In Vivo, Transfection, Reporter Assay, In Situ Hybridization, Sequencing, Software

Journal: Cerebellum (London, England)

Article Title: Sonic Hedgehog Agonist Protects Against Complex Neonatal Cerebellar Injury

doi: 10.1007/s12311-017-0895-0

Figure Lengend Snippet: The HIF pathway plays a role in maintaining CGNP proliferation, and overactivation in CGNPs result in prednisolone-mediated cerebellar hypoplasia. a Right, schematic diagram showing HIF pathway. Left, schematic of cerebellar circuit highlighting CGNP-specific Cre recombination (red). EGL external granule layer, PL Purkinje cell layer. b Representative images of CGNPs in the external granule layer (EGL) and CGNs in the internal granule layer (IGL) with absence or presence of HIF1α (red). Insert, mitotic cells positive for PH3 (green) in the EGL. Nuclei counterstained with DAPI (blue). Scale bar, 50 μm. c P11 animals show increased expression of the HIF target BNIP3 in homozygous floxed animals only. Representative lobule 8 of cerebellar vermis. EGL external granule layer, PL Purkinje cell layer, IGL internal granule layer. Scale bar, 50 μm. d Quantification of cerebellar size at P2. n.s. , no significant difference. e Quantification of PH3+ cells in EGL at P2. Math1Cre ; Vhl ( fl /+) = 27.17 ± 0.437 cells/mm 2 , Math1Cre ; Vhl ( fl / fl ) = 23.16 ± 1.40 cells/mm 2 . For quantification, n ≥ 3 per experiments, * p < 0.05, Student’s t test. f Representative images of lobule 6 in P22 brains receiving Pred administration from P3 to P11. Nuclei are counterstained with DAPI (blue) to visualize IGL. Scale bar, 50 μm. g Quantification of IGL cross-sectional area in P22 transgenic mice. Math1Cre ; Vhl ( fl /+) = 2.49 ± 0.264 mm 2 ( n = 3), Math1Cre ; Vhl ( fl / fl ) = 2.34 ± 0.103 mm 2 ( n = 4), Math1Cre ; Vhl ( fl /+) + Pred = 2.44 ± 0.257 mm 2 ( n = 7), Math1Cre ; Vhl ( fl / fl ) + Pred = 1.57 ± 0.190 mm 2 ( n = 6). ** p < 0.01, ANOVA with Tukey’s post-hoc correction. For quantification, n ≥ 3 experiments per condition. h Transfection of HIF1α overexpressing vector in primary CGNP cultures, and representative Western for HIF1α and cyclin D1 (CCD1), with β-Actin used for normalization. i Primary CGNP cultures from the Gli - Luciferase reporter mouse line were transfected with HIF1α construct and assayed for luciferase activity 24 h later. Values depicted as relative to signal intensity in control condition. Ctrl = 1 ± 0.15 arbitrary units (au), Ctrl + Shh n = 1.99 ± 0.098 au, HIF1a = 0.179 ± 0.0311 au, HIF1a + Shh n = 0.51 ± 0.018 au, Piggyback = 0.961 ± 0.092 au, Piggyback + Shh n = 1.77 ± 0.054 au. n = 3 per condition. * p > 0.05, Student’s t test

Article Snippet: For primary antibodies, we used PH3 (mouse monoclonal, Cell Signaling), Calbindin (mouse monoclonal or rabbit polyclonal, Swant), Cleaved Caspase 3 (rabbit polyclonal, Cell Signaling), NeuN (mouse monoclonal, Millipore), Iba1 (rabbit polyclonal, Wako), HIF1a (rabbit polyclonal, Cayman Chemicals), BNIP3 (rabbit polyclonal, Cell Signaling), and Cre (rabbit polyclonal, Millipore).

Techniques: Expressing, Transgenic Assay, Transfection, Plasmid Preparation, Western Blot, Luciferase, Construct, Activity Assay

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b , e , f Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( a , b ) or EPC ( e , f ) cells. The cells were collected at 36 h ( a , e ) or 48 h ( b , f ) post-transfection. c , d , g , h Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( c , d ) or EPC ( g , h ) cells for 24 h, followed by infection with GCRV for another 24 h ( c , g ) or 36 h ( d , h ). i – l Expression of irf3 , irf7 , and tbk1 mRNA after bmp8a knockdown in ZFL cells. The cells were collected at 36 h ( i ) and 48 h ( j ) post-knockdown or at 24 h ( k ) and 36 h ( l ) post-infected with GCRV. m , o Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( m ) or EPC ( o ) cells. The cells were collected at 36 or 48 h post-transfection for Immunoblot analysis. n , p Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( n ) or EPC ( p ) cells for 24 h, followed by infection with GCRV for another 24 or 36 h. q , r Immunoblot analysis of phosphorylated (p-) TBK1 and IRF3 after bmp8a knockdown in ZFL cells. The cells were collected at 36 and 48 h post-knockdown or at 24 and 36 h post-infected with GCRV. s – u EPC cells were cotransfected with IFN-φ1pro-luc (200 ng, s ), IFN-φ3pro-luc (200 ng, t ) or EPC IFNpro-luc (200 ng, u ), and bmp8a (100 ng) together with each of the dominant negative plasmids including tbk1–K38M (100 ng), irf3DN (100 ng) and irf7DN (100 ng). At 48 h post-transfection, the cells were collected for luciferase assays. Renilla luciferase was used as the internal control. v – y Expression of irf3 , irf7 , and tbk1 mRNA in the liver, kidney, intestine, and spleen from WT or bmp8a −/− zebrafish injected i.p. with 50 µl of GCRV (10 8 TCID 50 per ml). The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell post hoc tests for comparison of multiple groups. All data were presented as mean ± SD (** p < 0.01, *** p < 0.001).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Western Blot, Dominant Negative Mutation, Luciferase, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b Expression of alk2 , alk3 , alk6a , bmpr2a , bmpr2b , actr2a , and actr2b mRNA in ZFL cells stimulated with poly(I:C) (2 μg/ml, a ) or GCRV (5 × 10 4 TCID 50 per ml, b ) for 48 h. c – e Expression of ifnφ1 ( c ) and ifnφ3 ( d ) mRNA in ZFL cells or EPC ifn ( e ) in EPC cells which were transfected with 2 μg of alk2, alk3, alk6a, bmpr2a, bmpr2b, actr2a, actr2b or empty vector for 48 h. f , h Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of pcDNA3.1-alk6a or empty vector in ZFL ( f ) or EPC ( h ) cells for 48 h. g , i Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of of pcDNA3.1-alk6a or empty vector in ZFL ( g ) or EPC ( i ) cells for 24 h, followed by infection with GCRV for another 36 h. j Schematic drawing of the alk6a-ΔGS mutation that the GS domain of Alk6a was deleted. k , m Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( k ) or EPC ( m ) cells for 48 h. l , n Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( l ) or EPC ( n ) cells for 24 h, followed by infection with GCRV for another 36 h. The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell posthoc tests for comparison of multiple groups. All data were presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, ns means no significant difference).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: Upon virus infection, the transcriptions of bmp8a are activated through the Jak-Stat1 pathway. The Bmp8a binds to BMP type I receptor Alk6a, promoting phosphorylation of Tbk1 and Irf3 to induce the expression of Ifn through p38 MAPK pathway.

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Infection, Expressing

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Oligonucleotide sequences used in this study.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Primers used for plasmids construction.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: miR-146b-3p targets AKT1 and MDFIC and downregulates PI3K/AKT pathway activity. ( a ) Relative mRNA expression of AKT1 and MDFIC in QM-7 cells after overexpression or inhibition of miR-146b-3p. ( b ) The phosphorylation levels of AKT and AKT1 and the protein expression of AKT1 after transfecting miR-146b-3p mimic or inhibitor in CPMs. ( c ) Gray value analysis of protein bands in (b). ( d ) Dual-luciferase report assay performed after co-transfecting the wild type or mutant 3′UTR of AKT1 with miR-146b-3p mimic or mimic NC in DF-1 cells. ( e ) Dual-luciferase report assay performed after co-transfecting the wild type or mutant 3′UTR of MDFIC with miR-146b-3p mimic or mimic NC in DF-1 cells. ( f ) Relative mRNA expression of the cell differentiation-related genes after co-transfection. ( g ) Relative mRNA expression of the cell apoptosis-related genes after co-transfection. ( h ) Cell cycle analysis of QM-7 cells after co-transfection. Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Activity Assay, Expressing, Over Expression, Inhibition, Luciferase, Mutagenesis, Cell Differentiation, Cotransfection, Cell Cycle Assay, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: AKT1 and MDFIC promote myoblast proliferation. ( a ) Cell cycle analysis of CPMs after overexpression or inhibition of AKT1 . ( b ) Cell cycle analysis of CPMs after overexpression or inhibition of MDFIC . ( c ) Relative mRNA expression of the cell cycle-related genes after overexpression or inhibition of AKT1 in CPMs. ( d ) Relative mRNA expression of the cell cycle-related genes after overexpression or inhibition of MDFIC in CPMs. ( e ) EdU staining of CPMs after overexpression or inhibition of AKT1 . ( f ) The fold change of proliferation rates of CPMs with pcDNA3.1- AKT1 or si- AKT1 . ( g ) EdU staining of CPMs after overexpression or inhibition of MDFIC . ( h ) The fold change of proliferation rates of CPMs with pcDNA3.1- MDFIC or si- MDFIC . Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Cell Cycle Assay, Over Expression, Inhibition, Expressing, Staining, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Both AKT1 and MDFIC promote myoblast differentiation, while suppress myoblast apoptosis. ( a ) Relative expression of AKT1 in Xinghua chicken leg muscle from E11 to E18. ( b ) Relative expression of MDFIC in Xinghua chicken leg muscle from E11 to E18. ( c ) Relative mRNA expression of AKT1 during CPMs induced differentiation. ( d ) Relative mRNA expression of MDFIC during CPMs-induced differentiation. ( e , f ) Relative mRNA expression of the cell differentiation-related genes after overexpression or inhibition of AKT1 or MDFIC in CPMs. ( g , i ) Immunofluorescence of MyHC after overexpression or knockdown of AKT1 and MDFIC in CPMs. ( h , j ) Comparison of the area of myotubes described in ( g , i ). Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Expressing, Cell Differentiation, Over Expression, Inhibition, Immunofluorescence, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: AKT1 shows an inhibitory effect on myoblast apoptosis and so does MDFIC . ( a , b ) Flow cytometry analysis of Annexin V-FITC and PI dual staining detecting the apoptosis of CPMs after transfection of pcDNA3.1- AKT1 or si- AKT1 . ( c ) Relative mRNA expression of the cell apoptosis-related genes after transfection of pcDNA3.1- AKT1 or si- AKT1 in CPMs. ( d ) Relative mRNA expression of the cell apoptosis-related genes after transfection of pcDNA3.1- MDFIC or si- MDFIC in CPMs. ( e , f ) The protein levels of MyHC, cleaved-caspase 8, and cleaved-caspase 9 after the overexpression or silence of AKT1 and MDFIC in CPMs. ( g , h ) Gray value analysis of protein bands in ( e , f ). Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Flow Cytometry, Staining, Transfection, Expressing, Over Expression, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Model of miR-146b-3p mediated regulatory mechanism in myoblast proliferation, differentiation, and apoptosis. In simple terms, miR-146b-3p downregulates the expression of AKT1 and MDFIC by targeting the 3′UTR of their mRNA. Both the phosphorylation of AKT1 and AKT can trigger the activation of PI3K/AKT pathway, thereby promoting the expression of cell cycle-related genes and myoblast differentiation-related genes and suppress cell apoptosis-related genes, which in turn facilitate cell proliferation and differentiation and inhibit apoptosis. In addition, the expression of MDFIC can also lead to the same effect.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Expressing, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Prostaglandin E2 induces FOXP3 gene expression and T regulatory cell function in human CD4+ T cells.

doi: 10.4049/jimmunol.175.3.1483

Figure Lengend Snippet: FIGURE 3. PGE2 induces FOXP3 mRNA expression in both CD4CD25 T reg cells and CD4CD25 T cells. Purified CD4CD25 T reg cells (A) and CD4CD25 T cells (B) were incubated without () or with () PGE2 (26 M) for 24 h. C, Alternatively, CD4CD25 T cells were cultured for 24 h with medium alone or in tumor supernatant from COX-2 S (with or without anti-PGE2 Ab (10 g/ml) or mouse IgG control Ab (10 g/ml)), COX-2 AS, or CV-transfected H157. T cell FOXP3 mRNA expression was quantified after 48-h activation with plate-bound anti-CD3 Ab by real-time PCR as described in Materials and Methods. In CD4CD25 T reg cells treated with or without PGE2 (A), the fold increase in FOXP3 mRNA was relative to FOXP3 mRNA expression in CD4CD25. In PGE2-treated CD4CD25 cells (B), the fold change in FOXP3 mRNA was relative to that in untreated CD4CD25 cells. In CD4CD25 T cells cultured with gene-modified H157 supernatant (C), the fold change in FOXP3 was relative to that in CD4CD25 cells in medium alone. Results are expressed as the mean (SE) of one experiment of at least three performed in triplicate from different donors. Asterisks indicate statistical significance compared with the control value.

Article Snippet: FOXP3 was immunodetected with 1/5000 rabbit anti-human FOXP3 polyclonal Ab (Abcam), followed by incubation with 1/500 HRP-conjugated donkey antirabbit Ig (Santa Cruz Biotechnology).

Techniques: Expressing, Incubation, Cell Culture, Control, Transfection, Activation Assay, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Prostaglandin E2 induces FOXP3 gene expression and T regulatory cell function in human CD4+ T cells.

doi: 10.4049/jimmunol.175.3.1483

Figure Lengend Snippet: FIGURE 4. PGE2 enhances FOXP3 mRNA expression, gene transcription, and protein. A, Jurkat T cells were activated for 18 h as indicated. FOXP3 mRNA expression was quantified by real-time PCR as described in Materials and Methods. B, Top panel, FOXP3 protein was analyzed by Western blot after stimulation for 18 h as indicated. B, Bottom panel, Densitometric analysis of FOXP3 expression of the Western blot. One representative result of two independent experiments performed is shown. C, PGE2 induces FOXP3 promoter activity. Jurkat T cells cotransfected with FOXP3 promoter and firefly luciferase reporter gene constructs (FOXP3-SubD) or with Renilla luciferase gene (CV) were stimulated for 18 h as indicated. FOXP3 promoter expression was analyzed by a dual luciferase reporter assay system, and relative luciferase activity was expressed as the ratio of firefly to Renilla luciferase. A representative experiment of three performed is shown.

Article Snippet: FOXP3 was immunodetected with 1/5000 rabbit anti-human FOXP3 polyclonal Ab (Abcam), followed by incubation with 1/500 HRP-conjugated donkey antirabbit Ig (Santa Cruz Biotechnology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Construct, Reporter Assay

Journal: Computational and Mathematical Methods in Medicine

Article Title: miR-152-3p Affects the Progression of Colon Cancer via the KLF4/IFITM3 Axis

doi: 10.1155/2020/8209504

Figure Lengend Snippet: The overexpression of KLF4 mediates the IFITM3 expression to regulate colon cancer cell proliferation and apoptosis. (a) The expression of KLF4 and IFITM3 in the normal cell line CCD-18Co and colon cell lines HT29, HCT116, and SW480 was detected by qRT-PCR. (b) Western blot was carried out for the protein examination of KLF4 and IFITM3 in cells transfected with oe-NC+pre-NC, oe-KLF4+pre-NC, and oe-KLF4+pre-IFITM3. (c) CCK-8, (d) colony formation assay, and (e) flow cytometry were performed to determine cell viability, colony-forming ability, and cell apoptosis (∗ means p < 0.05).

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase- (HRP-) labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Primary antibodies included KLF4 rabbit polyclonal antibody (ab215036, 1 : 1000, Abcam, Cambridge, UK), IFITM3 rabbit polyclonal antibody (ab109429, 1 : 1000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1 : 2500, Abcam, Cambridge, UK).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Flow Cytometry

Journal: Computational and Mathematical Methods in Medicine

Article Title: miR-152-3p Affects the Progression of Colon Cancer via the KLF4/IFITM3 Axis

doi: 10.1155/2020/8209504

Figure Lengend Snippet: miR-152-3p targets KLF4 and decreases its expression. (a) Binding sites of miR-152-3p on KLF4 3′UTR were predicted by the bioinformatics method. (b) Dual-luciferase assay was done for the validation of the targeting relationship between miR-152-3p and KLF4. (c) Western blot was conducted to test the protein expression of KLF4 and IFITM3 in each treatment group (∗ means p < 0.05).

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase- (HRP-) labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Primary antibodies included KLF4 rabbit polyclonal antibody (ab215036, 1 : 1000, Abcam, Cambridge, UK), IFITM3 rabbit polyclonal antibody (ab109429, 1 : 1000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1 : 2500, Abcam, Cambridge, UK).

Techniques: Expressing, Binding Assay, Luciferase, Western Blot

Journal: Computational and Mathematical Methods in Medicine

Article Title: miR-152-3p Affects the Progression of Colon Cancer via the KLF4/IFITM3 Axis

doi: 10.1155/2020/8209504

Figure Lengend Snippet: miR-152-3p affects colon cancer cell proliferation and apoptosis via the KLF4/IFITM3 axis. (a) Western blot was carried out to determine the protein expression of KLF4 and IFITM3 in cells transfected with inhibitor NC+si-NC, miR-152-3p inhibitor+si-NC, and miR-152-3p inhibitor+si-KLF4. (b) CCK-8, (c) colony formation assay, and (d) flow cytometry were conducted to assay cell viability, colony-forming ability, and apoptosis in each treatment group (∗ means p < 0.05).

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase- (HRP-) labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Primary antibodies included KLF4 rabbit polyclonal antibody (ab215036, 1 : 1000, Abcam, Cambridge, UK), IFITM3 rabbit polyclonal antibody (ab109429, 1 : 1000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1 : 2500, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Transfection, CCK-8 Assay, Colony Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation

doi: 10.3389/fimmu.2020.576658

Figure Lengend Snippet: Emission of immunogenic cell death markers induced by combined adenoviral p14ARF + IFNβ gene transfer. SK-MEL-147 cells transduced as previously described, incubated for 48h h before cells and supernatants were collected for ICD assays. (A) Calreticulin exposure was assessed by flow cytometry after specific antibody staining. Representative dot plots and a bar graph showing the mean and standard deviation from three independent tests with three technical replicates each. (B) Supernatant from the same cultures were collected and evaluated for ATP secretion using a luciferase-based assay (RLU, relative light units). Data represent the mean and standard deviation from at least three independent experiments. (C) Detection of secreted IFNβ protein from cell supernatant by ELISA. Data represent the mean and standard deviation from at least three independent experiments. For both (A–C) , statistical analyses were performed using one-way ANOVA test followed by the Bonferroni post-test. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Article Snippet: For the evaluation of calreticulin exposure through flow cytometry, cells were probed with rabbit anti-calreticulin antibody (Novus Biologicals, Littleton, CO, USA), followed by the Alexa488-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific).

Techniques: Incubation, Flow Cytometry, Staining, Standard Deviation, Luciferase, Enzyme-linked Immunosorbent Assay