Journal: Molecular Cell

Article Title: Conserved Pseudoknots in lncRNA MEG3 Are Essential for Stimulation of the p53 Pathway

doi: 10.1016/j.molcel.2019.07.025

Figure Lengend Snippet: Functional Importance of MEG3 Structural Motifs (A–C) p53-dependent luciferase assays using the p53Luc reporter on MEG3 variants, individual exons, and domains (A); on D2-D3 mutants (B); and on selected H11 mutants (C). Construct D5 is indicated in parenthesis because it is more than 10,000-fold less expressed than v1. Expression levels of all other constructs are reported in Figure S5 O. Error bars indicate SEM of at least 3 experiments. Asterisks indicate a significant difference in relative luciferase signal with respect to v1 based on one-way ANOVA statistical tests in GraphPad ( ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001). (D) Sequences used to disrupt the H11 stem (H11-5′mut, red nucleotides) and corresponding compensatory mutations (H11-comp, green nucleotides). (E) qRT-PCR and western blot analysis of p53 upregulation by v1, v9, H11LpA, and G 370 C. (F) qRT-PCR analysis measuring upregulation of p53 target genes (BAX, p21, GADD45A, GDF15, and NOXA) by v1, v9, H11LpA, and G 370 C. Representative western blots for BAX and p21 are reported on the right (the endogenous signal for GADD45A, GDF15, and NOXA is too low in our system for accurate quantification). (E and F) Error bars indicate SEM of 2 biological replicas, each performed in technical triplicates, and the black line indicates that the right and left parts of the images were manually joined because they were separated from each other in the raw image of the blot. Asterisks indicate significant variation with respect to control (CTRL) based on one-way ANOVA statistical tests in GraphPad ( ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001). (G) RNA immunoprecipitation using the DO1 anti-p53 antibody for cells transfected with the indicated constructs. Control samples using unspecific immunoglobulin G (IgG) produced negligible amplification and are not plotted. Values are reported as percent input. Error bars indicate SEM of 4 biological replicas, each performed in technical duplicates. Asterisks indicate significant variations in the amounts of immunoprecipitated control RNAs (GAPDH and RNR1) with respect to the target RNA (v1, H11LpA, or DINO) based on unpaired parametric t tests in GraphPad ( ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001). (H) Pull-down of p53 (detected by western blot) using in vitro -transcribed and biotinylated v1 and H11LpA and non-biotinylated v1 (CTRL). Values are reported as percent of input. Error bars indicate SEM of 3 biological replicas. Asterisks indicate significant variation of p53 pulled down by v1 and H11LpA with respect to CTRL based on one-way ANOVA statistical tests in GraphPad ( ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001).

Article Snippet: anti BAX (D2D) Mouse IgG1 antibody , Santa Cruz Biotechnology, Inc. (Texas, USA) , Cat#sc-20067; RRID: AB_626726.

Techniques: Functional Assay, Luciferase, Construct, Expressing, Quantitative RT-PCR, Western Blot, Control, RNA Immunoprecipitation, Transfection, Produced, Amplification, Immunoprecipitation, In Vitro

Journal: Molecular Cell

Article Title: Conserved Pseudoknots in lncRNA MEG3 Are Essential for Stimulation of the p53 Pathway

doi: 10.1016/j.molcel.2019.07.025

Figure Lengend Snippet:

Article Snippet: anti BAX (D2D) Mouse IgG1 antibody , Santa Cruz Biotechnology, Inc. (Texas, USA) , Cat#sc-20067; RRID: AB_626726.

Techniques: Virus, Recombinant, Modification, Reverse Transcription, Blocking Assay, Flow Cytometry, Staining, cDNA Synthesis, Ex Vivo, In Vivo, Plasmid Preparation, Luciferase, Control, Software

Journal: Veterinary Research

Article Title: TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus

doi: 10.1186/s13567-024-01407-6

Figure Lengend Snippet: ASFV infection regulated coagulation and related factors in vivo . A Evolution of rectal temperature, B clinical score in animals (6 animals) IM infected with 10 2.5 HAD 50 of ASFV HLJ/18. C viremia, viremia values are expressed as copies/mL, sensitivity of virus detection: > 1000 copies/mL, and D mortality. Dpi: days post-infection. E Sketch of the coagulation activation process. F ASFV-positive serum samples from each pig at the indicated time points were collected for detection of F10 and G F10 activity. H ASFV-positive serum samples from each pig at the indicated time points were collected for detection of F2 and I D2D by ELISA. Statistical data were analysed by t test analysis of variance (* p < 0.05, ** p < 0.01, and *** p < 0.001). All the data are expressed as the mean ± SD.

Article Snippet: The pig coagulation F10 ELISA kit, pig coagulation factor II ELISA kit, and D-dimer ELISA kit were purchased from Cusabio Company (Wuhan, China).

Techniques: Infection, Coagulation, In Vivo, Virus, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus

doi: 10.1186/s13567-024-01407-6

Figure Lengend Snippet: ASFV-encoded proteins regulated F10 expression and activity . A Several encoded proteins expressed in the baculovirus expression system were added to Huh7 cells at the same concentration for 24 h prior to supernatant and cell lysate collection. The mixtures were detected for F10 activity. B Huh7 cells were transfected with plasmids expressing ASFV-encoded proteins, including pK205R, pE199L, pEP153R, p30, p54, and p72, and then, the cells and supernatants were collected for detection of F10 expression by western blotting, C and secretory F10 expression by ELISA. For all figures, the experiments were repeated at least three times with similar results. The data are presented as the mean ± SD from one single experiment. Statistical significance was determined by Student’s t test (* p < 0.05).

Article Snippet: The pig coagulation F10 ELISA kit, pig coagulation factor II ELISA kit, and D-dimer ELISA kit were purchased from Cusabio Company (Wuhan, China).

Techniques: Expressing, Activity Assay, Concentration Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus

doi: 10.1186/s13567-024-01407-6

Figure Lengend Snippet: Functional domain of p72 . A Schematic of truncated p72 expression. B Confocal microscopy of truncated proteins in Huh7 cells; the target proteins are stained red. DAPI was used to stain the sections blue. Bar, 20 μm. C Truncated plasmids containing amino acids 1–150, 151–422, and 423–646, D 151–450, E and 423–646 of p72 with an HA tag at the C-terminus were transfected (0.5, 1, or 2 μg) into Huh7 cells. At 24 h post-transfection, the cell lysate was collected and subjected to western blot analysis, and the F cell supernatant was collected for detection of secretory F10 expression via ELISA. G Mutant plasmid construction schematic. The sequences of the six p72 mutants ranged from 423–450 aa, and every 10 amino acids were randomly mutated to alanine, which were named 101, 102 (423–432 aa), 201, 202 (433–442 aa), 301, and 302 (443–452 aa). H Confocal microscopy of mutant proteins in Huh7 cells. The target proteins are stained green. DAPI was used to stain the sections blue. Bar, 20 μm. I The mutant plasmids were transfected into Huh7 cells at increasing doses (0.5, 1, and 2 μg) . At 24 h post-transfection, the cells were lysed with lysis buffer, and 15 μg of each sample was subjected to western blotting. The band intensities were normalized to those of GAPDH via ImageJ software. The data are expressed as the mean intensity ratio ± SD of F10 to GAPDH. The experiment was performed in triplicate, and images from three independent experiments were plotted. J The cell supernatant was also collected for detection of secretory F10 expression via ELISA. Statistical data were analysed by t test variance (* p < 0.05, ns represents nonsignificant p > 0.05).

Article Snippet: The pig coagulation F10 ELISA kit, pig coagulation factor II ELISA kit, and D-dimer ELISA kit were purchased from Cusabio Company (Wuhan, China).

Techniques: Functional Assay, Expressing, Confocal Microscopy, Staining, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Plasmid Preparation, Lysis, Software