11031 system  (Promega)

Journal: Frontiers in Microbiology

Article Title: Combination of fluorescent reagents with 2-(4-aminophenyl) benzothiazole and safranin O was useful for analysis of spore structure, indicating the diversity of Bacillales species spores

doi: 10.3389/fmicb.2025.1603957

Figure Lengend Snippet: Phase contrast and fluorescence microscopic images of Bacillales spores stained with APBT and safranin O. cells of Bacillus subtilis (A) , B. licheniformis (B) , Niallia circulans (C) , Brevibacillus brevis (D) , Lysinibacillus sphaericus (E) , and Paenibacillus polymyxa (F) were suspended in a mixture of 2-(4-aminophenyl) benzothiazole (APBT) and safranin O and observed using phase contrast and fluorescence microscopy. From left to right: phase contrast images, APBT fluorescence, green fluorescence of safranin O, red fluorescence of safranin O, and merged images. Blue represents APBT fluorescence, while green and red indicate the green and red fluorescence of safranin O, respectively. Arrows indicate vegetative cells (VCs), mother cells (MCs), and forespores (FSs), and arrowheads indicate mature spores (MSs) in phase-contrast microscopy images. The scale bar represents 5 μm.

Article Snippet: In addition to B. subtilis , the following Bacillales spore-forming bacteria were used: Niallia circulans NBRC 13629, B. licheniformis ATCC 14580, Brevibacillus brevis NBRC 100599, Lysinibacillus sphaericus NBRC 3526, and Paenibacillus polymyxa NBRC15309.

Techniques: Fluorescence, Staining, Microscopy

Journal: Cell reports

Article Title: CPSF1 inhibition promotes widespread use of intergenic polyadenylation sites and impairs glycolysis in prostate cancer cells

doi: 10.1016/j.celrep.2024.115211

Figure Lengend Snippet: (A) Indicated mRNAs encoding factors in the CPSF, CstF, CFIm, and CFIIm complexes were tested for differential expression between metastatic CRPC (mCRPC) and primary prostate cancer specimens in public microarray datasets. (B–E) For each indicated gene, patients in the top vs. bottom quartiles of gene expression were tested for associations with progression-free survival using TCGA RNA-seq data. (F–I) Western blot of CPSF1 (top) and Tubulin (bottom) in LNCaP (F), LNCaP95 (G), 22Rv1 (H), and RWPE-1 (I) cells infected with lentivirus encoding shRNAs targeting CPSF1 (CPSF1 sh1 and CPSF1 sh2) or a non-targeting shRNA (CTRL sh). (J–M) Growth of LNCaP, LNCaP95, 22Rv1, and RWPE-1 cells infected as in (F)–(I). Cell growth was measured on days 1 and 6 post-seeding using crystal violet staining. Data are mean ± 95% CI. Significance was assessed by unpaired two-sided t tests. Abs, absorbance.

Article Snippet: Antibodies were purchased from Santa Cruz for western blot detection of CPSF1, (G-10, sc-166281, 1:100) and tubulin (B-5-1-2, sc-23948, 1:3000), Proteintech for western blot detection of NUDT21 (66335-1-Ig, 1:500) and PCF11 (23540-1-AP, 1:500), Fortis Life Sciences for western blot detection of CPSF6 (A301-356A, 1:1,000) and Origene for western blot detection of GPI (TA501171, 1:500).

Techniques: Quantitative Proteomics, Microarray, Gene Expression, RNA Sequencing, Western Blot, Infection, shRNA, Staining

Journal: Cell reports

Article Title: CPSF1 inhibition promotes widespread use of intergenic polyadenylation sites and impairs glycolysis in prostate cancer cells

doi: 10.1016/j.celrep.2024.115211

Figure Lengend Snippet: (A) RNA-seq experimental design for knockdown of CPSF1 with two independent shRNAs targeting CPSF1 (shCPSF1-1 and -2) or control shRNA (shCTRL). (B–D) Volcano plots showing global downregulation and upregulation of genes upon CPSF1 knockdown in LNCaP, LNCaP95, and 22Rv1 cells. Genes in volcano plots are colored blue or red based on whether they are downregulated or upregulated with false discovery rate (FDR) < 0.05. (E–G) Normalized enrichment scores for all 50 MSigDB Hallmark signatures derived from gene set enrichment analysis (GSEA) in LNCaP, LNCaP95, and 22Rv1 cells (differential expression in shCTRL vs. shCPSF1). Hallmark signatures are colored red if the FDR < 0.05. (H–J) ECAR output of CPSF1 shRNA- or CTRL shRNA-infected LNCaP, LNCaP95, and 22Rv1 cells. (K–M) Basal and compensatory glycolysis of CPSF1 shRNA- or CTRL shRNA-infected LNCaP, LNCaP95, and 22Rv1 cells. ECAR, extracellular acidification rate; GlycoPER, glycolytic proton efflux rate. Data are mean ± 95% CI. Significance was assessed by unpaired two-sided t tests.

Article Snippet: Antibodies were purchased from Santa Cruz for western blot detection of CPSF1, (G-10, sc-166281, 1:100) and tubulin (B-5-1-2, sc-23948, 1:3000), Proteintech for western blot detection of NUDT21 (66335-1-Ig, 1:500) and PCF11 (23540-1-AP, 1:500), Fortis Life Sciences for western blot detection of CPSF6 (A301-356A, 1:1,000) and Origene for western blot detection of GPI (TA501171, 1:500).

Techniques: RNA Sequencing, Knockdown, Control, shRNA, Derivative Assay, Quantitative Proteomics, Infection

Journal: Cell reports

Article Title: CPSF1 inhibition promotes widespread use of intergenic polyadenylation sites and impairs glycolysis in prostate cancer cells

doi: 10.1016/j.celrep.2024.115211

Figure Lengend Snippet: (A) PAC-seq experimental design for knockdown of CPSF1 with two independent shRNAs targeting CPSF1 (shCPSF1-1 and −2) or control shRNA (shCTRL). (B–D) Diagrams illustrating how changes in PAC-seq reads from CPSF1 shRNA and CTRL shRNA were quantified in intronic regions, CDS regions, or downstream of annotated 3′ UTR sequence (10 kb downstream region). (E–M) Volcano plots showing higher or lower PAS usage in intronic, CDS, or 10 kb downstream regions in CPSF1 shRNA vs. CTRL shRNA in LNCaP cells (E–G), LNCaP95 cells (H–J), and 22Rv1 cells (K–M). PASs in volcano plots are colored blue or red based on whether they are upregulated or downregulated with FDR < 0.05. PAS, poly(A) site; CDS, coding sequence.

Article Snippet: Antibodies were purchased from Santa Cruz for western blot detection of CPSF1, (G-10, sc-166281, 1:100) and tubulin (B-5-1-2, sc-23948, 1:3000), Proteintech for western blot detection of NUDT21 (66335-1-Ig, 1:500) and PCF11 (23540-1-AP, 1:500), Fortis Life Sciences for western blot detection of CPSF6 (A301-356A, 1:1,000) and Origene for western blot detection of GPI (TA501171, 1:500).

Techniques: Knockdown, Control, shRNA, Sequencing

Journal: Cell reports

Article Title: CPSF1 inhibition promotes widespread use of intergenic polyadenylation sites and impairs glycolysis in prostate cancer cells

doi: 10.1016/j.celrep.2024.115211

Figure Lengend Snippet: (A and B) Example plot and diagram illustrating the 3′ UTR length changes (PAC score) and expression changes that occur in each quadrant. (C–E) Density plots illustrating the 10 kb downstream PAC score and RNA-seq differential expression of genes in LNCaP, LNCaP95, and 22Rv1 cells infected with CPSF1 shRNA. Numbers represent the amount of genes represented in each quadrant. (F–H) Coverage of PAC-seq reads for representative quadrant I genes UGT2B17 in LNCaP cells, GPI in LNCaP95 cells, and ALDH3A2 in 22Rv1 cells. Diagrams on the bottom illustrate the effect of CPSF1 knockdown on 3′ UTR length for each indicated transcript. Red lines denote poly(A) sites called by DPAC analysis. (I and J) Pearson correlation analysis of the QI gene signature (avg gene Z score) to CPSF1 Z score in TCGA (I) and SU2C (J) patient datasets. (K) The QI gene signature (avg gene Z score) plotted in normal and primary prostate cancer patient data from TCGA. (L and M) Pearson correlation analysis of the HALLMARK_GLYCOLYSIS gene set (avg gene Z score) to CPSF1 Z score (L) and QI gene signature (avg gene Z score) (M) in the SU2C patient dataset. Significance was assessed by unpaired two-sided t tests. Q1, quadrant I; cPAS, canonical poly(A) site; dsPAS, extended poly(A) site; avg, average.

Article Snippet: Antibodies were purchased from Santa Cruz for western blot detection of CPSF1, (G-10, sc-166281, 1:100) and tubulin (B-5-1-2, sc-23948, 1:3000), Proteintech for western blot detection of NUDT21 (66335-1-Ig, 1:500) and PCF11 (23540-1-AP, 1:500), Fortis Life Sciences for western blot detection of CPSF6 (A301-356A, 1:1,000) and Origene for western blot detection of GPI (TA501171, 1:500).

Techniques: Expressing, RNA Sequencing, Quantitative Proteomics, Infection, shRNA, Knockdown

Journal: Cell reports

Article Title: CPSF1 inhibition promotes widespread use of intergenic polyadenylation sites and impairs glycolysis in prostate cancer cells

doi: 10.1016/j.celrep.2024.115211

Figure Lengend Snippet: (A) Illustration of the stepwise conversion of glycolysis to pyruvate. (B–D) Knockdown of CPSF1 decreases GPI mRNA (B) and protein levels (C) but shows no effect on nascent GPI mRNA (D). (E and F) qPCR (E) and PCR (F) of the extended 3′ UTR of GPI mRNA in CPSF1 shRNA-infected cells. (G) Disrupting the GPI canonical poly(A) site causes a 3′ UTR extension of GPI and decreased expression. (H and I) Disrupting the GPI canonical poly(A) site lowers ECAR output (H) and basal and compensatory glycolysis (I). (J) Disrupting the downstream poly(A) site of GPI in CPSF1-knockdown cells inhibits the extension of the GPI 3′ UTR. (K–M) Coverage of PAC-seq reads for PFKM (K), ALDOA (L), and PGK1 (M). Red lines denote poly(A) sites called by DPAC analysis. (N–P) qPCR validation of the 3′ UTR extension and decreased expression for PFKM (N), ALDOA (O), and PGK1 (P). (Q and R) CTRL shRNA and CPSF1 shRNA cells were treated with actinomycin D (Act-D) and the percentage of remaining mRNA relative to vehicle was quantified by qPCR. For half-life calculations, 6 h post-Act-D treatment was defined as time “0” because no decay was observed from 6 h of treatment. All experiments were performed in LNCaP95 cells. Data are mean with SD. Significance was assessed by unpaired two-sided t tests. RT, reverse transcriptase; cPAS, canonical poly(A) site; dsPAS, downstream poly(A) site; PAM, poly(A) morpholino; t 1/2 , half-life (hours).

Article Snippet: Antibodies were purchased from Santa Cruz for western blot detection of CPSF1, (G-10, sc-166281, 1:100) and tubulin (B-5-1-2, sc-23948, 1:3000), Proteintech for western blot detection of NUDT21 (66335-1-Ig, 1:500) and PCF11 (23540-1-AP, 1:500), Fortis Life Sciences for western blot detection of CPSF6 (A301-356A, 1:1,000) and Origene for western blot detection of GPI (TA501171, 1:500).

Techniques: Knockdown, shRNA, Infection, Expressing, Biomarker Discovery, Reverse Transcription

Journal: Cell reports

Article Title: CPSF1 inhibition promotes widespread use of intergenic polyadenylation sites and impairs glycolysis in prostate cancer cells

doi: 10.1016/j.celrep.2024.115211

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies were purchased from Santa Cruz for western blot detection of CPSF1, (G-10, sc-166281, 1:100) and tubulin (B-5-1-2, sc-23948, 1:3000), Proteintech for western blot detection of NUDT21 (66335-1-Ig, 1:500) and PCF11 (23540-1-AP, 1:500), Fortis Life Sciences for western blot detection of CPSF6 (A301-356A, 1:1,000) and Origene for western blot detection of GPI (TA501171, 1:500).

Techniques: Recombinant, shRNA, Over Expression, Plasmid Preparation, Cloning, Virus, Software