Journal: iScience

Article Title: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal

doi: 10.1016/j.isci.2022.104804

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-CstF77 antibody (G-5) , Santa Cruz Biotechnology , Cat#sc-376575; RRID: AB_11151026.

Techniques: Recombinant, Cell Viability Assay, shRNA, Control, Plasmid Preparation, Software

Journal: Computational and Structural Biotechnology Journal

Article Title: Alternative polyadenylation regulates the translation of metabolic and inflammation-related proteins in adipose tissue of gestational diabetes mellitus

doi: 10.1016/j.csbj.2024.03.013

Figure Lengend Snippet: The decrease in APA-related trans-acting factors causes 3'UTR elongation and reduced protein translation. (a) Differential protein levels of APA-related trans-acting factors in different group comparisons, with 10 out of 23 factors not detected. (b) Pearson correlation coefficients between the protein levels of trans-acting factors and the mean PDUI in OM adipose tissues show a strong correlation between trans-acting factors and APA. (c) Pearson correlation coefficients of protein levels between trans-acting factors (CSTF3 and PPP1CB) and other genes in OM adipose tissue indicate a strong positive correlation between trans-acting factors and protein translation levels. (d) Cell population distribution of CSTF3 (top) and PPP1CB (bottom). (e) Expression levels of CSTF3 (top) and PPP1CB (bottom) in various cell populations. (f) Experimental verification of protein expression levels of CSTF3 and PPP1CB in OM adipose tissue. (g) The knockdown efficiency of CSTF3 and PPP1CB in TDMs, HUVECs and ADSCs. (h) qRT-PCR validations for lengthening and shortening APA isoforms of LRRC25 and RAI14. Results are presented as the relative proportion of 3'UTR lengthening isoforms in different groups. Fig. S1 Distribution and functional enrichment of APA events across different groups. (a) Heatmap of dynamic APA events in adipose tissue. (b) PCA plots for samples originating from the same tissue source. (c) Scatter plots of mean PDUI values for genes in different group comparisons, with a cutoff value of ∆PDUI = ± 0.1. (d) Functional enrichment networks of significantly different APA events in each group comparison, with clusters of functions connected by edges. The top 20 clusters by P-value are shown. Fig. S2 RNA-seq tracks and PDUI levels for representative genes in different group comparisons: GSC vs NSC group (a), NOM vs NSC group (b), and GOM vs GSC group (c). Fig. S3 Distribution of genes in different group comparisons: PCA plots of mRNA (a) and protein (b) from the GDM and normal groups. DEmRNA expression heatmaps (c) and volcano plots (d), DEP expression heatmaps (e), and volcano plots (f). The cut-off values were │FC│≥ 1.2 and adj. P < 0.01 for DEmRNAs, │FC│≥ 1.2 and adj. P < 0.05 for DEPs. Fig. S4 Gene distribution differences between the transcriptome and proteome. (a) Distribution of Pearson correlation coefficients for protein-mRNA matched genes with the proportion of genes above and below ± 0.5 indicated. (b-d) mRNA and protein expression levels of representative genes in the GSC vs NSC (b), NOM vs NSC (c), and GOM vs GSC (d) groups. Fig. S5 Partial results from the snRNA-seq analysis: Distribution of cells from GOM and NOM groups in tSNE projection (a) and heatmap of cell markers for different cell populations (b). Additional material information. Additional file 1: Table S1 The clinic characteristics of GDM patients and controls. Additional file 2: Table S2 The primers for qRT-PCR and overexpression vectors and the sequence of siRNA. Additional file 3: Table S3 The dynamic APA events calculated by Dapars algorithm. Additional file 4: Fig. S1 Distribution and functional enrichment of APA events across different groups. Additional file 5: Fig. S2 RNA-seq tracks and PDUI levels for representative genes in different group comparisons. Additional file 6: Fig. S3 Distribution of DEGs in different group comparisons. Additional file 7: Table S4 The transcriptomics and proteomics data analysis in different group comparisons. Additional file 8: Table S5 The functional enrichment analysis of DEPs in different group comparisons. Additional file 9: Fig. S4 Gene distribution differences between the transcriptome and proteome. Additional file 10: Table S6 The protein-per-mRNA FC ratio analysis for mRNA-protein matched genes in different group comparisons. Additional file 11: Table S7 The GO biological process enrichment results for potentially APA-regulated genes. Additional file 12: Table S8 Predicted binding miRNAs between proximal and distal PAS of mRNA-Protein matched genes and their relation with GDM. Additional file 13: Fig. S5 Partial results from the snRNA-seq analysis.

Article Snippet: The primary antibodies used in Western blot included LRRC25 (Bioss, bs-12339R), CSTF3 (Proteintech, 24290–1-AP) and PPP1CB (Proteintech, 10140–2-AP).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Functional Assay, Comparison, RNA Sequencing, Over Expression, Sequencing, Binding Assay

Journal: Gene

Article Title: Polyadenylation Site-specific Differences in the Activity of the Neuronal βCstF-64 Protein in PC-12 Cells

doi: 10.1016/j.gene.2013.08.007

Figure Lengend Snippet: βCstF-64 interacts with CstF-77 in PC-12 cells. A) PC-12 cells were seeded onto 10 cm dishes and transfected with 10 μg of 3×FLAG (lanes 1–3), 3×FLAG-CstF-64 (lanes 4–6) or 3×FLAG-βCstF-64 (lanes 7–9) using Lipofectamine 2000. Cells were harvested 48 hours post-transfection. An aliquot of cell lysate (5%) was used as “Total extract” control (upper and lower panels, lanes 1, 4, and 7) while the remaining was added to anti-FLAG antibody coupled to agarose resin and incubated overnight at 4°C. Beads were centrifuged and an aliquot of the supernatant (5%) was used to analyze “unbound” proteins (upper and lower panels, lanes 3, 6, and 9). After extensive washing with NET-2 buffer, “bound” proteins were eluted by boiling in SDS loading buffer. The eluted proteins (50%) were resolved by SDS-PAGE, transferred to nitrocellulose membrane and subjected to immunoblot analysis (IB) using anti-FLAG (upper panel) or anti-CstF-77 antibodies (lower panel). The apparent molecular masses of the 3×FLAG-CstF-64 and βCstF-64 proteins and CstF-77 are indicated on left. B, C. Exogenously expressed CstF-64 and βCstF-64 proteins localize to the nucleus. PC-12 cells were grown on coverslips in 24-well plates and transfected with FLAG-MS2-CstF-64 (panel A) or FLAG-MS2-βCstF-64 using Lipofectamine 2000. After 48 hours, cells were fixed with paraformaldehyde, permeabilized with Triton-X-100 and incubated with anti-FLAG antibody for two hours. Secondary antibody conjugated to Alexa fluor 488 (green) was added and slides containing these coverslips were subsequently used for photomicrography. The images shown above are an overlay of the phase contrast (red) with the fluorescent signal emanating from the FLAG-CstF-64 and βCstF-64 proteins. Arrows indicate anti-FLAG staining of FLAG-CstF-64 (panel B) or FLAG-βCstF-64 (panel C), predominantly in nuclei; arrowheads denote less intense cytoplasmic staining, indicating a paucity of FLAG-CstF-64 (panel B) or FLAG-βCstF-64 (panel C) in cytoplasm.

Article Snippet: 2.2 Antibodies The following antibodies were used in this study: anti-α-tubulin and anti-FLAG antibodies (Sigma, St. Louis, MO); mouse monoclonal anti-CstF-64 (3A7, [ 17 ]); rabbit anti-βCstF-64 [ 15 ]; mouse monoclonal anti-CstF-77 (Abnova, Walnut, CA, catalog #H00001479-M01); and rabbit polyclonal anti-CstF-77 (Bethyl, Montgomery, TX, catalog #A301-096A).

Techniques: Transfection, Control, Incubation, SDS Page, Membrane, Western Blot, Staining