Journal: bioRxiv

Article Title: “Targeting LARP1 Enhances Carboplatin Sensitivity and Suppresses Tumor Growth in Endometrial Cancer”

doi: 10.64898/2026.03.22.713473

Figure Lengend Snippet: (A, B) Kaplan–Meier plot showing that overexpression of E2F1 is associated with shorter overall survival (A) and progression-free interval (B) in endometrial cancer cohort. (C, D) Kaplan–Meier plot showing that co-overexpression of LARP1 and E2F1 is associated with shorter overall survival (C) and progression-free interval (D) in endometrial cancer cohort. (E) Spearman correlation analysis shows a correlation between LARP1 and E2F1 in endometrial cancer patients.

Article Snippet: Cells were then incubated overnight at 4 °C with the following primary antibodies: LARP1 (Cat. No. sc-515873; 1:200 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) and E2F1 (Cat. No. 3742S; 1:400 dilution; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression

Journal: bioRxiv

Article Title: “Targeting LARP1 Enhances Carboplatin Sensitivity and Suppresses Tumor Growth in Endometrial Cancer”

doi: 10.64898/2026.03.22.713473

Figure Lengend Snippet: (A, B) Immunoblot analysis showing the protein expression of LARP1 and E2F1 after transfecting ISHI (A) and HEC-1A (B) cells with control or LARP1 siRNA. β-actin was used as a loading control. (C) Images representing immunofluorescence staining of LARP1 (green) and E2F1 (red) in HEC-1A cells after transfection with control or LARP1 siRNA. DAPI was used as a counter stain. Scale bar = 50 µm.

Article Snippet: Cells were then incubated overnight at 4 °C with the following primary antibodies: LARP1 (Cat. No. sc-515873; 1:200 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) and E2F1 (Cat. No. 3742S; 1:400 dilution; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Transfection

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: The pRb–E2F pathway regulates RNA splicing of E2F target genes. ( A ) Differential changes in splicing between WT and E2F1 Cr HCT116 cells, treated with DMSO or 1 μM T1-44 for 48 h are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event across all samples, as compared to the WT E2F1 HCT116 cells treated with DMSO (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. Black boxes indicate splice events that uniquely occur upon T1-44 treatment, only in the presence of WT E2F1. These data were generated from three independent biological samples. Venn diagram showing the overlap between statistically significant differential splicing events (FDR < 0.05) (AS) in each treatment, as compared to WT E2F1 HCT116 cells treated with DMSO. These data were generated from three independent biological samples. ( C ) A representative immunoblot displaying input protein levels of E2F1 and symmetric dimethylation (SDMe). Actin served as a loading control. ( D ) The bar chart displays the breakdown of statistically significant (FDR < 0.05) differential splicing events observed in each of the indicated treatments, as compared to WT E2F1 HCT116 cells treated with DMSO. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. and . ( E ) Annotation of genes which undergo splice events that uniquely occur upon T1-44 treatment in the presence of WT E2F1 (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle, stress responses, and DNA damage are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( F ) Differential changes in splicing between WT and Rb Cr MCF7 cells, treated with DMSO or 1 μM T1-44 for 48 h are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event across all samples, as compared to the WT Rb MCF7 cells treated with DMSO (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. Black boxes indicate splice events that uniquely occur upon T1-44 treatment, only in the presence of WT Rb. These data were generated from three independent biological samples. Venn diagram showing the overlap between statistically significant differential splicing events (FDR < 0.05) (AS) in each treatment, as compared to WT Rb MCF7 cells treated with DMSO. These data were generated from three independent biological samples. ( H ) A representative immunoblot displaying input protein levels of Rb and SDMe. GAPDH served as a loading control. ( I ) The bar chart displays the breakdown of statistically significant (FDR < 0.05) differential splicing events observed in each of the indicated treatments, as compared to WT Rb MCF7 cells treated with DMSO. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. and . ( J ) Annotation of genes which undergo splice events that uniquely occur upon T1-44 treatment in the presence of WT Rb (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle, stress responses, and DNA damage are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( K ) WT E2F1 and E2F1 Cr HCT116 cells treated for 48 h with 1 μM T1-44 or DMSO as indicated. An RT-PCR was performed to measure the inclusion of VCAN exon 7, MDM1 exon 4, METTL6 exon 3, or REV3L exon 3 in RNA transcripts from the cells. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. A diagram indicating the exon (boxed in grey) and intron (black lines) structure of each gene around the skipped exon (boxed in yellow) of interest is included. The splicing that gives rise to the exon included and excluded transcripts is also displayed, with specific primer pairs used in QPCR shown as blue arrows. A representative immunoblot is included to display input protein levels of E2F1 and SDMe. Actin was used as a loading control. (biological repeats: n = 4 for VCAN and METTL6, n = 3 for REV3L , and n = 8 for MDM1 ). See also . ( L ) A ChIP assay performed on WT E2F1 or E2F1 Cr HCT116 cells. Recruitment of E2F1 to the promoter regions of MDM1 and VCAN was tested. CDC6 acted as a positive control. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs. An immunoblot is included to display input protein levels of E2F1 and SDMe. Actin was used as a loading control (biological repeats: n = 3 for MDM1, VCAN , and CDC6 ).

Article Snippet: Cleared extracts were then divided equally into tubes and mixed with 3 μg of E2F1 specific IgG (KH-95, sc-251, Santa Cruz, Dallas, USA; or clone G10, Argonaut Therapeutics, Oxford, UK) overnight.

Techniques: Generated, Western Blot, Control, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: The pRb–E2F pathway regulates separate transcription and splicing gene networks. ( A ) Differential gene expression between WT and E2F1 Cr HCT116 cells, treated with DMSO or 1 μM T1-44 for 48 h, are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG across all samples, as compared to the WT E2F1 HCT116 cells treated with DMSO (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. Black boxes indicate DEGs that uniquely occur upon T1-44 treatment, only in the presence of WT E2F1. These data were generated from three independent biological samples. See also . ( B ) A Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) in each treatment, as compared to WT E2F1 HCT116 cells treated with DMSO. See also . ( C ) Annotation of genes which only undergo differential expression upon T1-44 treatment in the presence of WT E2F1 (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( D ) Differential gene expression between WT and Rb Cr MCF7 cells, treated with DMSO or 1 μM T1-44 for 48 h, are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG across all samples, as compared to the WT Rb MCF7 cells treated with DMSO (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. Black boxes indicate DEGs that uniquely occur upon T1-44 treatment, only in the presence of WT Rb. These data were generated from three independent biological samples. See also Supplementary Fig. S3E. ( E ) A Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) (DEGs) in each treatment, as compared to WT Rb MCF7 cells treated with DMSO. See also . ( F ) Annotation of genes which only undergo differential expression upon T1-44 treatment in the presence of WT Rb (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( G ) Venn diagrams displaying the overlap of total genes from the E2F1 Cr and Rb Cr RNA-seq datasets that score either as significantly differentially expressed (DEGs: padj < 0.05; log2FC > 0.58), significantly differentially spliced (AS: FDR < 0.05), or fall into both categories. These data were derived from the analyses in Figs and . ( H ) Venn diagrams showing the overlap between the total list of statistically significant DEGs (padj < 0.05, log2FC > 0.58), or the total list of statistically significant differentially spliced genes (AS: FDR < 0.05) in each of the indicated RNA-sequencing datasets, as compared to their corresponding wild-type cell lines.

Article Snippet: Cleared extracts were then divided equally into tubes and mixed with 3 μg of E2F1 specific IgG (KH-95, sc-251, Santa Cruz, Dallas, USA; or clone G10, Argonaut Therapeutics, Oxford, UK) overnight.

Techniques: Gene Expression, Expressing, Generated, Quantitative Proteomics, RNA Sequencing, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: Cell cycle regulated alternative splicing and transcriptional events dependent on E2F1. ( A ) On the left, a representative flow cytometry profile for wild-type (WT) E2F1 and E2F1 Cr HCT116 cells synchronized at the G1/S boundary with a double thymidine block (0 h timepoint), or released from the block to progress through S phase (3 h) into G2/M (6 h), and back into a subsequent G1 (9 and 12 h) are displayed. An RT-PCR was performed at each timepoint to measure the inclusion of MDM1 exon 4 in RNA transcripts. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. A representative immunoblot is included to display input protein levels of Rb, phosphorylated Rb and E2F1. GAPDH was used as a loading control ( n = 4 biological repeats). See also . ( B ) WT E2F1 and E2F1 Cr HCT116 cells were treated with 1 mM hydroxyurea (HU) for 24 h to synchronize cells in early S phase. Hydroxyurea was then washed out and cells were allowed to progress through S phase for the indicated number of hours. Alternatively, cells were treated with 20 μM etoposide (Etop) for 48 h where indicated, and an RT-PCR was performed to measure the inclusion of MDM1 exon 4 in RNA transcripts. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 4 biological repeats). See also . ( C ) A representative immunoblot to display input protein levels of Rb, phosphorylated Rb, E2F1, HNRNPC, and SRSF2 for the experiments described in Fig. . GAPDH was used as a loading control. ( D ) Differential changes in splicing between WT E2F1 and E2F1 Cr HCT116 cells synchronized in G1/S (0 h) or G2/M (6 h) are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event in cells at 6 h, as compared to the same cells at 0 h (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. These data were generated from four independent biological samples. A representative immunoblot is included to display the input protein levels for E2F1. GAPDH was used as a loading control. ( E ) Venn diagram showing the overlap between statistically significant differentially spliced genes (AS: FDR < 0.05) between the 6 h (G2/M) and 0 h (G1/S) timepoints, in WT E2F1 and E2F1 Cr HCT116 cells. These data were derived from the analysis in Fig. . ( F ) Annotation of genes which undergo splice events that uniquely occur in WT E2F1 cells between the 6 and 0 h timepoints (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle and DNA damage/repair are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( G ) The bar charts display the breakdown of statistically significant (FDR < 0.05) differential splicing events observed between each of the indicated treatments. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. . ( H ) Differential changes in gene expression between WT E2F1 and E2F1 Cr HCT116 cells synchronized in G1/S (0 h) or G2/M (6 h) are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG in cells at 6 h, as compared to the same cells at 0 h (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. These data were generated from four independent biological samples. See also . ( I ) Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) between the 6 h (G2/M) and 0 h (G1/S) timepoints, in WT E2F1 and E2F1 Cr HCT116 cells. These data were derived from the analysis in Fig. . ( J ) Annotation of genes which undergo differential expression only in WT E2F1 cells between the 6 and 0 h timepoints (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( K ) Venn diagram displaying the overlap of total genes from the RNA-seq experiment in Fig. and H, that score either as significantly differentially expressed (DEGs: padj < 0.05, log2FC > 0.58), significantly differentially spliced (AS: FDR < 0.05), or fall into both categories.

Article Snippet: Cleared extracts were then divided equally into tubes and mixed with 3 μg of E2F1 specific IgG (KH-95, sc-251, Santa Cruz, Dallas, USA; or clone G10, Argonaut Therapeutics, Oxford, UK) overnight.

Techniques: Alternative Splicing, Flow Cytometry, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Generated, Derivative Assay, Gene Expression, Expressing, Quantitative Proteomics, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: Confirmation of cell cycle regulated alternative splicing and transcriptional events in cells. ( A ) RNA from WT E2F1 and E2F1 Cr cells either in G1/S (0 h) or G2/M (6 h) was used in an RT-PCR experiment to monitor the inclusion of exon 3 in TRPT1 , exons 2 and 3 in TRMT1 , exon 5 in DEPDC4 , and exon 17 in SORBS1 , each identified as being significantly differentially spliced in the analysis performed in Fig. . Displayed is the mean inclusion/exclusion ratio with SD. A diagram indicating the exon (boxed in grey) and intron (black lines) structure of each gene around the skipped exon (boxed in yellow) of interest is included. The splicing that gives rise to the exon included and excluded transcripts is also displayed, with specific primer pairs used in QPCR shown as blue arrows. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. (biological repeats: n = 4 for TRMT1 ex2 + 3 and SORBS1 ex17, n = 5 for TRPT1 exon 3 and DEPDC4 exon 5). ( B ) RNA from WT E2F1 and E2F1 Cr cells either in G1/S (0 h) or G2/M (6 h) was used in an RT-PCR experiment to monitor the expression of genes ( THBS1, RCAN1 and IL18R1 ) identified as significantly differentially expressed in the RNA-sequencing experiment performed in Fig. . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 6 biological repeats). ( C ) A ChIP assay performed on wild-type E2F1 HCT116 cells. Recruitment of E2F1 to the promoter regions of the indicated differentially spliced genes ( TRPT1, TRMT1, DEPDC4 , and SORBS1 ) and DEGs ( THBS1, RCAN1 , and IL18R1 ) was tested. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs ( n = 3 biological repeats).

Article Snippet: Cleared extracts were then divided equally into tubes and mixed with 3 μg of E2F1 specific IgG (KH-95, sc-251, Santa Cruz, Dallas, USA; or clone G10, Argonaut Therapeutics, Oxford, UK) overnight.

Techniques: Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: RNA splicing factors associate with the E2F complex and are recruited to AS genes. ( A ) A representative immunoblot displaying the immunoprecipitation of E2F1 from WT HCT116 cells treated with 1 μM T1-44 or DMSO for 48 h where indicated. E2F1 Cr HCT116 cells were used in the E2F1 immunoprecipitation as a control. Rb is displayed on the immunoblots as a known interactor for E2F1, and SDMe is used as a control for T1-44 activity. GAPDH is presented as a loading control. The immunoprecipitated material was used in a downstream mass spectrometry analysis of the E2F1 interactome ( n = 3 bioligical repeats). ( B ) Volcano plots displaying log2 fold change (log2FC) and –log10 P -values for relative intensities of interacting proteins in E2F1 immunoprecipitates performed in T1-44 treated or untreated WT cells, as compared to immunoprecipitations performed in the control E2F1 Cr cell line. Red colour represents interacting proteins enriched in the WT E2F1 immunoprecipitates, whilst blue colour represents under-enriched proteins. Grey colour represents proteins that fell below the fold change or statistical cut-off applied ( P < 0.05, log2FC > 1). Marked on the figure are known E2F1 interacting proteins (Rb, E2F1, DP1, and DP2) and proteins implicated in RNA splicing and processing. These data were derived from the mass spectrometry experiment performed in Fig. ( n = 3 biological repeats). ( C ) Functional protein association networks for proteins identified as E2F1 interactors from mass spectrometry analysis performed on DMSO treated (i), or T1-44 treated cells (ii) were generated using STRING. Proteins that were enriched in E2F1 immunoprecipitations performed in WT cells, as compared to E2F1 Cr cells (fold change > 2), at a statistically significant level ( P < 0.05) were included. The edges indicate both functional and physical protein associations, with line thickness indicating the strength of data support. MCL clustering of proteins was performed. This figure was generated using the data from Fig. . ( D ) An immunoprecipitation experiment was performed in WT E2F1 HCT116 cells using the indicated antibodies against E2F1 (KH95 and G10 antibodies) or control IgG. Interacting SRSF2 and HNRNPC was detected using specific antibodies. Input protein levels are also displayed ( n = 2 bioligical repeats). See also . ( E ) An RIP assay was performed in (i) wild-type (WT) E2F1 HCT116 cells treated with 1 μM T1-44 or DMSO for 48 h as indicated, (ii) or in WT E2F1 and E2F1 Cr HCT116 cells. Anti-HNRNPC (HNC), -SRSF2 (SR2), -HNRNPH1 (HNH), or control IgG was used to immunoprecipitate the indicated splicing factors and bound RNA. Recruitment of splicing factors to the indicated exon regions (exon 3, 4, and 5) of MDM1 are shown. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs. (iii) A diagram of the exon (boxed in grey) and intron (black lines) structure around the MDM1 skipped exon 4 (boxed in yellow) is displayed. Specific exon–intron flanking primer pairs used in the RIP analysis are indicated by blue arrows. (iv) A representative immunoblot is included to display input protein levels of E2F1, HNRNPC, SRSF2, HNRNPH1, and SDMe. α-Tubulin was used as a loading control ( n = 3 biological repeats).

Article Snippet: Cleared extracts were then divided equally into tubes and mixed with 3 μg of E2F1 specific IgG (KH-95, sc-251, Santa Cruz, Dallas, USA; or clone G10, Argonaut Therapeutics, Oxford, UK) overnight.

Techniques: Western Blot, Immunoprecipitation, Control, Activity Assay, Mass Spectrometry, Derivative Assay, Functional Assay, Generated

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: RNA splicing factors contribute to E2F-pathway dependent alternative splicing. ( A ) WT E2F1 HCT116 cells were transfected with control siRNA (siC) or siRNA against SRSF2 (siSR2) prior to treatment with 1 μM T1-44 or DMSO where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of SRSF2 . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). WT E2F1 and E2F1 Cr HCT116 cells were transfected with control siRNA (siC) or siRNA against SRSF2 (siSR2) where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of SRSF2 . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). ( C ) A representative immunoblot displaying input protein levels of E2F1 and SRSF2 for the experiments described in Fig. and . SDMe levels are also displayed and GAPDH served as a loading control. ( D ) WT E2F1 HCT116 cells were transfected with control siRNA (siC) or siRNA against HNRNPC (siHNC) prior to treatment with 1 μM T1-44 or DMSO where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of HNRNPC . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). WT E2F1 and E2F1 Cr HCT116 cells were transfected with control siRNA (siC) or siRNA against HNRNPC (siHNC) where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of HNRNPC . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). ( F ) A representative immunoblot displaying input protein levels of E2F1 and HNRNPC for the experiments described in Fig. and . SDMe levels are also displayed and GAPDH served as a loading control. ( G ) Model diagram indicating that the pRb–E2F complex, in concert with PRMT5 activity, regulates cell cycle dependent expression of E2F target genes both at the level of transcriptional control (i), and at the level of AS (ii). E2F target gene networks regulated by transcription or AS tend to be mutually exclusive. AS regulation is achieved in part through interactions between the pRb–E2F complex with RNA splicing factors, including SRSF2 and HNRNPC. E2F and PRMT5 activity regulate the recruitment of these splicing factors to mRNA around AS exons (ii).

Article Snippet: Cleared extracts were then divided equally into tubes and mixed with 3 μg of E2F1 specific IgG (KH-95, sc-251, Santa Cruz, Dallas, USA; or clone G10, Argonaut Therapeutics, Oxford, UK) overnight.

Techniques: Alternative Splicing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Activity Assay