Journal: PLoS ONE

Article Title: Comprehensive Analysis of Preeclampsia-Associated DNA Methylation in the Placenta

doi: 10.1371/journal.pone.0107318

Figure Lengend Snippet: DNA Methylation Changes in Early Onset Preeclampsia (EOPET) Versus Normal Controls (Norm).

Article Snippet: cg04502814 , 29 , 43 , 7.73E-06 , 0.004750353 , 5 , SEPP1.

Techniques: DNA Methylation Assay

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: MiR-183-5p promotes VSMC proliferation and migration by reducing FKBPL levels. (A) Venn diagram analysis of miRNAs that hsa_circ_0001402 might interact with. (B-C) Western blot analysis of FKBPL, p21, PCNA, MMP9, and MMP2 after 48 h of transfection of miR-Control or miR-183-5p mimic in HASMCs. Data are shown as mean ± SD ( n = 3, * p < 0.05, ** p < 0.01 vs. miR-Control group, two-tailed paired t -test). (D) The binding site between the CDS region of FKBPL or Fkbpl mRNA and miR-183-5p. (E) Dual-luciferase reporter gene analysis of the interaction between miR-183-5p and FKBPL . WT: wild-type; MUT: mutant. Data are shown as mean ± SD ( n = 6, *** p < 0.001, two-tailed unpaired t -test). (F) EdU incorporation analysis of DNA synthesis after 48 h of transfection of miR-Control or miR-183-5p mimic in HASMCs. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents HASMCs with DNA synthesis. The scale bar is 10 µm. (G) The proportion of EdU-positive HASMCs. Data are shown as mean ± SD ( n = 5, * p < 0.05 vs. miR-Control group, two-tailed unpaired t -test with Welch's correction). (H) CCK-8 analysis of cell viability of HASMCs transfected with miR-Control or miR-183-5p mimic. Data are shown as mean ± SD ( n = 6, *** p < 0.001 vs. miR-Control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (I) Migration analysis of MOVAS cells transfected with miR-Control or miR-183-5p mimic. The scale bar is 500 µm. (J) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, *** p < 0.001, two-tailed unpaired t -test). (K-L) Western blot analysis of FKBPL, p21, PCNA, MMP9, and MMP2 after 48 h of transfection of miR-Control or miR-183-5p inhibitor in MOVAS cells. Data are shown as mean ± SD ( n = 4 or 3, ** p < 0.01 vs. inhibitor-miR-Control group, two-tailed paired t -test). (M) CCK-8 analysis of cell viability of MOVAS cells transfected with miR-Control or miR-183-5p inhibitor. Data are shown as mean ± SD ( n = 9, *** p < 0.001 vs. inhibitor-miR-Control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (N) EdU incorporation analysis of DNA synthesis after 48 h of transfection of miR-Control or miR-183-5p inhibitor in MOVAS cells. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents MOVAS cells with DNA synthesis. The scale bar is 10 µm. (O) The proportion of EdU-positive MOVAS cells. Data are shown as mean ± SD ( n = 5, *** p < 0.001 vs. inhibitor-miR-Control group, two-tailed unpaired t -test). (P) Migration analysis of MOVAS cells transfected with miR-Control or miR-183-5p inhibitor. The scale bar is 500 µm. (Q) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, ** p < 0.01, two-tailed unpaired t -test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Migration, Western Blot, Transfection, Control, Two Tailed Test, Binding Assay, Luciferase, Mutagenesis, DNA Synthesis, Fluorescence, CCK-8 Assay

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Overexpression of miR-183-5p aggravates neointimal hyperplasia by decreasing FKBPL and BECN1 levels. (A) The cross-sectional H&E staining images of the ligated mouse common carotid arteries after 21 days of treatment with Sham Control, miR-Control agomir, or miR-183-5p agomir. The scale bar is 100 µm. (B) Morphometric measurement of the intimal/media ratio in the mouse common carotid arterial sections ( n = 4 mice). Data are shown as mean ± SD (*** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (C) Immunofluorescence of FKBPL after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (D) The quantification of FKBPL levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control agomir group, two-tailed unpaired t -test). (E) Immunofluorescence of p21 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (F) The quantification of p21 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (G) Immunofluorescence of PCNA after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (H) The quantification of PCNA levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (I) Immunofluorescence of MMP9 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (J) The quantification of MMP9 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (K) Immunofluorescence of MMP2 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (L) The quantification of MMP2 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (M) Immunofluorescence of BECN1 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (N) The quantification of BECN1 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (O) Immunofluorescence of p62 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (P) The quantification of p62 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are presented as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (Q) Immunofluorescence of total LC3 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (R) The quantification of total LC3 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). Normal mouse IgG was used as a negative control. MFI: mean fluorescent intensity.

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Over Expression, Staining, Control, Immunofluorescence, In Situ, Two Tailed Test, Negative Control

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Silencing of miR-183-5p alleviates neointimal hyperplasia by increasing FKBPL and BECN1 levels. (A) The cross-sectional H&E staining images of the ligated mouse common carotid arteries after 21 days of treatment with Sham Control, miR-Control antagomir, or miR-183-5p antagomir. The scale bar is 100 µm. (B) Morphometric measurement of the intimal/media ratio in the mouse common carotid arterial sections ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (C) Immunofluorescence of FKBPL after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (D) The quantification of FKBPL levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (*** p < 0.001 vs. miR-Control antagomir group, two-tailed unpaired t -test). (E) Immunofluorescence of p21 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (F) The quantification of p21 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). (G) Immunofluorescence of PCNA after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (H) The quantification of PCNA levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control antagomir group, two-tailed unpaired t -test). (I) Immunofluorescence of MMP9 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (J) The quantification of MMP9 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control antagomir group, two-tailed unpaired t -test). (K) Immunofluorescence of MMP2 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (L) The quantification of MMP2 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). (M) Immunofluorescence of BECN1 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (N) The quantification of BECN1 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control antagomir group, two-tailed unpaired t -test). (O) Immunofluorescence of p62 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (P) The quantification of p62 levels in the ligated mouse common carotid arteries ( n = 6 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). (Q) Immunofluorescence of total LC3 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (R) The quantification of total LC3 levels in the ligated mouse common carotid arteries ( n = 6 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). Normal mouse IgG was used as a negative control. MFI: mean fluorescent intensity.

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Staining, Control, Immunofluorescence, In Situ, Two Tailed Test, Negative Control

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Overexpression of hsa_circ_0001402 increases FKBPL and BECN1 levels by sponging miR-183-5p. (A) qRT-PCR analysis of miR-183-5p expression in HASMCs transfected with the expression vector of hsa_circ_Control or hsa_circ_0001402 , normalized to U6 . Data are shown as mean ± SD ( n = 12, *** p < 0.001 vs. hsa_circ_Control group, two-tailed unpaired t -test). (B) The binding site between hsa_circ_0001402 and miR-183-5p. (C) Dual-luciferase reporter gene analysis of the interaction between hsa_circ_0001402 and miR-183-5p. Data are shown as mean ± SD [ n = 6, ** p < 0.01, two-tailed unpaired t -test (left), two-tailed Mann-Whitney U test (right)]. (D-E) Western blot analysis of PCNA, MMP9, and MMP2 in HASMCs co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. Data are shown as mean ± SD ( n = 4, * p < 0.05, ** p < 0.01 vs. control group, two-tailed paired t -test). (F) EdU incorporation analysis of DNA synthesis in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents MOVAS cells with DNA synthesis. The scale bar is 10 µm. (G) The proportion of EdU-positive MOVAS cells. Data are shown as mean ± SD ( n = 6, *** p < 0.001 vs. control group, two-tailed unpaired t -test). (H) CCK-8 analysis of cell viability of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic. Data are shown as mean ± SD ( n = 9, ** p < 0.01, *** p < 0.001 vs. control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (I) Migration analysis of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic. The scale bar is 500 µm. (J) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, * p < 0.05, ** p < 0.01, two-tailed unpaired t -test). (K-L) Western blot analysis of FKBPL and p21 after 48 h of transfection of the expression vector of hsa_circ_Control or hsa_circ_0001402 in HASMCs. Data are shown as mean ± SD ( n = 3, * p < 0.05 vs. hsa_circ_Control group, two-tailed paired t -test). (M) Overexpression of hsa_circ_0001402 competitively binds to miR-183-5p to reduce the latter's inhibition of FKBPL -WT luciferase activity. Data are shown as mean ± SD ( n = 6, *** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (N) EdU incorporation analysis of DNA synthesis in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either Control or Fkbpl siRNA for 48 h. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents MOVAS cells with DNA synthesis. The scale bar is 10 µm. (O) The proportion of EdU-positive MOVAS cells. Data are shown as mean ± SD ( n = 6, *** p < 0.001 vs. siRNA-Control group, two-tailed unpaired t -test). (P) CCK-8 analysis of cell viability of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either Control or Fkbpl siRNA. Data are shown as mean ± SD ( n = 9, *** p < 0.001 vs. siRNA-Control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (Q) Migration analysis of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either Control or Fkbpl siRNA. The scale bar is 500 µm. (R) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, ** p < 0.01, two-tailed unpaired t -test). (S-T) Western blot analysis of LC3 in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. Data are shown as mean ± SD ( n = 4, * p < 0.05, *** p < 0.001 vs. control group, two-tailed paired t -test). (U) TEM analysis of autolysosomes in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. The scale bar is 1 μm. (V) Quantify the number of autolysosomes. Data are shown as mean ± SD ( n = 6, * p < 0.05 vs. control group, two-tailed paired t -test). (W) Western blot analysis of BECN1 after 48 h of transfection of the expression vector of hsa_circ_Control or hsa_circ_0001402 in HASMCs. Data are shown as mean ± SD ( n = 3, * p < 0.05 vs. hsa_circ_Control group, two-tailed paired t -test). (X) Overexpression of hsa_circ_0001402 competitively binds to miR-183-5p to reduce the latter's inhibition of BECN1 -WT luciferase activity. Data are shown as mean ± SD ( n = 6, * p < 0.05, ** p < 0.01, Kruskal-Wallis test followed by Dunn's multiple comparisons test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Two Tailed Test, Binding Assay, Luciferase, MANN-WHITNEY, Western Blot, DNA Synthesis, Fluorescence, CCK-8 Assay, Migration, Inhibition, Activity Assay

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Overexpression of hsa_circ_0001402 alleviates neointimal hyperplasia by increasing FKBPL and BECN1 levels. (A) The cross-sectional H&E staining images of the ligated mouse common carotid arteries after 21 days of treatment with Sham Control, hsa_circ_Control lentivirus, or hsa_circ_0001402 lentivirus. The scale bar is 100 µm. (B) Morphometric measurement of the intimal/media ratio in the mouse common carotid arterial sections ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (C) Immunofluorescence of FKBPL after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (D) The quantification of FKBPL levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (E) Immunofluorescence of p21 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (F) The quantification of p21 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (G) Immunofluorescence of PCNA after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (H) The quantification of PCNA levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (I) Immunofluorescence of MMP9 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (J) The quantification of MMP9 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (K) Immunofluorescence of MMP2 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (L) The quantification of MMP2 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (M) Immunofluorescence of BECN1 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (N) The quantification of BECN1 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (O) Immunofluorescence of p62 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (P) The quantification of p62 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (Q) Immunofluorescence of total LC3 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (R) The quantification of total LC3 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). Normal mouse IgG was used as a negative control. MFI: mean fluorescent intensity.

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Over Expression, Staining, Control, Immunofluorescence, In Situ, Two Tailed Test, Negative Control

Journal: Nature

Article Title: Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

doi: 10.1038/nature13441

Figure Lengend Snippet: Figure 2 | Mutant IDH blocks hepatocyte differentiation by silencing HNF- 4a. a, Heat map of GSEA showing top-ranked gene sets distinguishing IDH1(R132C) or IDH2(R172K) from wild-type (WT) or empty vector (EV) control hepatoblasts (pairwise analysis; replicates for each condition; see Methods). NES, normalized enrichment score; NS, not significant. b, c, Hepatoblasts analysed by immunoblot (b) and qRT–PCR (c). d, e, Analysis of wild-type hepatoblasts expressing the indicated short hairpin (sh)RNAs (uncoated plates). CTL, control. d, Hepatocyte sphere formation. e, Proliferation of shRNA-expressing hepatoblast cells co-expressing EV or shRNA-resistant Hnf4a(1) complementary DNA. f–h, Control and R132C- expressing hepatoblasts co-expressing vector control (EV2) or HNF-4a, grown on uncoated plates. f, Hepatocyte sphere formation. g, Hepatocyte gene expression. h, Proliferation. Scale bars, 100mm (d), 250mm (f). *P , 0.05.

Article Snippet: Human wild-type IDH1 cDNA (NCBI RefSeq accession number NM_ 005896.3) was obtained from Origene and subcloned into pRetro-Puro using EcoRI and BamHI fragments.

Techniques: Mutagenesis, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Expressing, shRNA, Gene Expression

Journal: Virology

Article Title: Chinese hamster ovary cell lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for NPC1 expression.

doi: 10.1016/j.virol.2012.05.018

Figure Lengend Snippet: Fig. 6. Resistant cell lines have mutations in NPC1. (a) Schematic of NPC1 protein. Location of insertions in R1 (star) and R2 (circle), and deletion (triangle) and substitution (diamond) in L2 indicated in diagram at top and in sequence at bottom. (b) Expression of NPC in WT, M12, and resistant cells transduced with pLenti CMV NPC1 Hygro or pLenti CMV GFP Hygro. (c) Resistant cells stably expressing NPC1 or GFP were challenged with VSV EboGP mCherry and infection was quantitated by flow cytometry. Bars represent average of means from three separate experiments done in triplicate; errors bars represent standard error means.

Article Snippet: The rLuc gene was removed from VSV-XN2 rLuc by cleavage with MluI and NheI and replaced with the GP-KpnIjunction-XhoI-mCherry sequence. pLenti CMV NPC1 Hygro was constructed by removing the GFP gene from pLenti CMV GFP Hygro and inserting into its place the NPC1 cDNA (Origene). pSport6 Axl (clone ID 5205825) was from Open Biosystems. pcDNA3.1 furin and pcDNA3.1-ACE2 were previously described (Simmons et al., 2005; Wool-Lewis and Bates, 1999).

Techniques: Sequencing, Expressing, Transduction, Stable Transfection, Infection, Cytometry