pflag cmv 2 maml1  (Addgene inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: The sequence of the primers used for gene expression analysis

Article Snippet: After 24 h, cells were transfected with 1.2 µg/ml of H19-pFLAG-CMV-2 expression vector (cat. n° E7033, Sigma-Aldrich, USA), or empty pFLAG-CMV-2 as Negative Control (Sigma-Aldrich).

Techniques: Sequencing, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: The sequence of the biotin-labeled H19 probes used for RNA pull-down assays

Article Snippet: After 24 h, cells were transfected with 1.2 µg/ml of H19-pFLAG-CMV-2 expression vector (cat. n° E7033, Sigma-Aldrich, USA), or empty pFLAG-CMV-2 as Negative Control (Sigma-Aldrich).

Techniques: Sequencing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: RBFOX2 is involved in RAC1 alternative splicing in CRC cells. ( A - B - D , E - F - H ) QRT-PCR for the indicated mRNA in CRC cells, silenced for RBFOX2 with two different siRNA. Graphs show 2-ΔΔct calculated in silenced cells respect to relative controls (Normality test and t-test). ( C , F ) Western Blot and densitometric analyses for RAC1B in SW620 and HCT116 silenced for RBFOX2 and relative controls. For densitometric analysis data are represented as normalized OD. ( I - L ) qRT-PCR of the indicated genes in SW620 and HCT116 silenced for H19, the graphs represent the 2^- ΔΔ ct of the indicated calculated respect the expression in control cells. ( M ) Western Blot of RAC1b protein levels in CRC cells (SW620 and HCT116) in H19 silenced cells and relative control cells. Statistical analyses were performed using normality test and t-test, p-value is shown in th e graphs

Article Snippet: After 24 h, cells were transfected with 1.2 µg/ml of H19-pFLAG-CMV-2 expression vector (cat. n° E7033, Sigma-Aldrich, USA), or empty pFLAG-CMV-2 as Negative Control (Sigma-Aldrich).

Techniques: Alternative Splicing, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer

doi: 10.1186/s13046-024-03139-z

Figure Lengend Snippet: Overexpression of lncH19 upregulates RAC1B expression. ( A ) qRT-PCR to analyze H19 mRNA levels in CRC cells (SW620 and HCT116) transfected with lncH19 or empty vector. ( B ) qRT-PCR to analyze RAC1B mRNA levels in CRC cells (SW620 and HCT116) transfected with lncH19 or empty vector. ( C ) Western Blot for RAC1B in CRC cells (SW620 and HCT116), transfected with lncH19 or empty vector. lncH19 overexpression promotes RAC1B nuclear localization. ( D ) Representative confocal microscopy images of anti-RAC1b immunofluorescence showed nuclear localization of RAC1b in lncH19-overexpressing CRC cells, SW620 (upper panel) and HCT116 (lower panel). The analysis of the RAC1B nuclear signal is reported on the histogram. ( E ) Western blot analysis and densitometric analyses for RAC1B in nuclear protein fractions of CRC cells (SW620 and HCT116) overexpressing H19 cells and relative control cells. E-F) Overexpression of lncH19 upregulate CiclinD and c-Myc expression through RAC1B activity qRT-PCR for the indicated genes in SW620 ( F ) and HCT116 ( G ) transfected with pH19 or empty vector and treated or not with RAC1B inhibitor. Statistical analyses were performed using: normality test, one sample t-test to compare different conditions to pEmpty untreated cells, and Two-tails unpaired t-test to compare two groups *; p-value is shown in the graphs

Article Snippet: After 24 h, cells were transfected with 1.2 µg/ml of H19-pFLAG-CMV-2 expression vector (cat. n° E7033, Sigma-Aldrich, USA), or empty pFLAG-CMV-2 as Negative Control (Sigma-Aldrich).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Confocal Microscopy, Immunofluorescence, Control, Activity Assay