plpc vector  (Addgene inc)

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

Article Snippet: On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions.

Techniques: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test