plko 1 shscr  (Addgene inc)

Journal: Molecular Medicine Reports

Article Title: SLC7A1 , SGK1 and HMGB2 are overexpressed in cervical cancer tissues and the miR-23b-3p/HMGB2 axis regulates cell migration and invasion

doi: 10.3892/mmr.2025.13600

Figure Lengend Snippet: Target genes of miR-124-3p, miR-23b-3p and shared by both miRNAs. (A) The Venn diagram shows the target mRNAs of miR-124-3p and miR-23b-3p recorded in miRDB and TargetScan. (B) The table shows the name of the 136 genes predicted as targets shared by miR-124-3p and miR-23b-3p. miR, microRNA; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: As a negative control, mirVana miRNA Mimic Negative Control #1, Scrambled, (Invitrogen; Thermo Fisher Scientific, Inc.) was used.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: N-acetyltransferase 10 promotes glioblastoma malignancy via mRNA stabilization of jumonji and AT-rich interaction domain containing 2

doi: 10.1016/j.jbc.2025.108544

Figure Lengend Snippet: NAT10 regulates JARID2 mRNA stability by acetylation of 2320 cytidine residue in JARID2 CDS . A , RNA precipitation (RIP) assay using anti-NAT10 antibodies. Upper panel shows western blotting analysis of NAT10 in immune-precipitates by anti-NAT10 antibodies. Lower graph shows quantitative real-time RT-PCR analysis of NAT10-RIP using primers listed in . Values are the mean with S.D. (n = 3). B , difference in the stability of JARID2 mRNA between naive and NAT10 knockout (KO) U251 cells. The mRNA levels were normalized by ATP5E mRNA levels. Basal levels of expression (0 h; the time of the initiation of ActD treatment) was set at 1.0. Values show the mean with S.D. (n = 4). ∗; p < 0.05 significant difference between the two groups. ( F 1,17 = 5.936, p = 0.026; Two-way ANOVA with the Tukey-Kramer post hoc test). C , The reporter activity of JARID2 CDS::Luc in U251 cells transfected with shRNA against NAT10 . Control cells were transfected with scramble shRNA (shScramble). Schematic diagram of JARID2 CDS::Luc is shown in the top of panels. Values of firefly luciferase activity were normalized to renilla luciferase activity. Values show the mean with S.D. (n = 4). ∗; p < 0.05 significant difference between the two groups ( t 6 = 3.006, Student’s t test). D , the reporter activity of JARID2 3′-UTR::Luc in U251 cells transfected with shRNA against NAT10 . Schematic diagram of JARID2 3′-UTR::Luc is shown in the top of panel. Control cells were transfected with shScramble. Values of firefly luciferase activity were normalized to renilla luciferase activity. Values show the mean with S.D. (n = 4). ( t 6 = 1.670, Student’s t test). E , left panel shows Sanger sequencing of wild-type and mutated (T192 and L774 deletion) JARID2 CDS::Luc. Black arrowheads indicate the NAT10-mediated acetylation site identified from RedaC:T-seq ( GSE162043 ). Right graph shows the reporter activity of wild-type JARID2 CDS::Luc, JARID2 T192 del::Luc, JARID2 L774 Del::Luc, and JARID2 T192 + L774 del::Luc in U251 cells transfected with shRNA against NAT10 . Control cells were transfected with scramble shRNA. Luciferase activities of shScramble-transfected cells are set at 1.0. Values of firefly luciferase activity were normalized to renilla luciferase activity and show the mean with S.D. (n = 4). ∗∗; p < 0.01 significant difference between the two groups. ( F 7,24 = 46.719, p < 0.001, ANOVA with the Tukey-Kramer post hoc test). F, Left panel shows Sanger sequencing of wild-type and point mutated (2320C > A, T, or G) JARID2 CDS::Luc. Right graph shows the reporter activity of wild-type JARID2 CDS::Luc, JARID2 2320C > A::Luc, JARID2 2320C > T::Luc, and JARID2 2320 C > G::Luc in U251 cells transfected with shRNA against NAT10 . Control cells were transfected with scramble shRNA. Luciferase activities of shScramble-transfected cells are set at 1.0. Values of firefly luciferase activity were normalized to renilla luciferase activity and show the mean with S.D. (n = 4). ∗; p < 0.05 significant difference between the two groups. ( F 7,24 = 4.357, p = 0.003, ANOVA with the Tukey-Kramer post hoc test).

Article Snippet: Scramble shRNA (Scramble[shRNA#1]) and JARID2 shRNA (pLV[shRNA]-Puro-U6>hJARID2[shRNA#1]) expressing plasmid were provided by VectorBuilder.

Techniques: Residue, Western Blot, Quantitative RT-PCR, Knock-Out, Expressing, Activity Assay, Transfection, shRNA, Control, Luciferase, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: N-acetyltransferase 10 promotes glioblastoma malignancy via mRNA stabilization of jumonji and AT-rich interaction domain containing 2

doi: 10.1016/j.jbc.2025.108544

Figure Lengend Snippet: Contribution of JARID2 to the maintenance of stemness properties of GBM cells . A , decrease in the growth ability of U251 and A172 cells by downregulation of JARID2. Each upper panel show JARID2 protein levels in U251 and A172 cells transfected with shRNA against JARID2 or scramble shRNA (shScramble). Each below graph shows the cell viability of seeding day (day 0) was set at 1.0. Values show the mean with S.D. (n = 6). ∗∗; p < 0.01 significant difference from shScramble group at corresponding time points. ( F 9,50 = 1636.125 p < 0.001 for U251 cells, F 9,50 = 140.977 p < 0.001 for A172 cells, ANOVA with the Tukey-Kramer post hoc test). B , the spheroid formation ability of U251 and A172 cells transfected with shRNA against JARID2 or shScramble. Eash left panel shows a representative photograph of Hoechst33342-stained spheroids formed by shScramble- or shJARID2-transfected U251 and A172 cells. Right panel shows the number of spheroids and the distribution of their diameters. Values show the mean with S.D. (n = 5–6 for U251 cells, n = 8 for A172 cells). ∗∗; p < 0.01 significant difference between the two groups ( t = 11.897. for U251 cells, t = 3.412 for A172 cells, Welch’s t test). C , the invasion ability of U251 and A172 cells transfected with shRNA against JARID2 or shScramble. Microphotographs show invasion of cells into 3D collagen gel. D , the protein expression levels of SOX2, OCT4, and KLF4 in U251 cells transfected with shRNA against JARID2 or scramble shRNA (shScramble). Values of protein levels were normalized to p84 protein levels. Values show the mean with S.D. (n = 5). ∗∗; p < 0.01 significant difference between the two groups ( t 8 = 3.407 for SOX2, t 8 = 12.072 for KLF4, Student’s t test). E , the protein expression levels N-CADHERIN and VIMENTIN in shScramble- or shJARID2-transfected U251 cells. Values of protein levels were normalized to those of β-ACTIN levels. Values show the mean with S.D. (n = 5). ∗∗; p < 0.01 significant difference between the two groups ( t 8 = 9.389 for N-CADHERIN, t 8 = 6.029 for VIMENTIN, Student’s t test). F , decrease in the malignancy of U251 cells by downregulation of JARID2. shScramble or shJARID2 RNA expressing lentivirus transfected U251 cells were subcutaneously implanted in Balb/c-nude mice. Left graph shows Kaplan-Meier survival curves shScramble or shJARID2 RNA expressing lentivirus transfected U251 tumor-bearing mice (n = 7 for shScramble, n = 9 for shJARID2). Right graph shows the tumor volume of each individual mouse. ∗; p < 0.05 significant difference between the two groups (LogRank Holm-Sidak test).

Article Snippet: Scramble shRNA (Scramble[shRNA#1]) and JARID2 shRNA (pLV[shRNA]-Puro-U6>hJARID2[shRNA#1]) expressing plasmid were provided by VectorBuilder.

Techniques: Transfection, shRNA, Staining, Expressing