Journal: Advanced Science

Article Title: Pirin Inhibits FAS‐Mediated Apoptosis to Support Colorectal Cancer Survival

doi: 10.1002/advs.202301476

Figure Lengend Snippet:

Article Snippet: CCG‐203971 , Selleck Chemicals , S8469.

Techniques: Virus, Recombinant, Cell Isolation, Sequencing, Software

Journal: bioRxiv

Article Title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

doi: 10.1101/2023.09.08.556810

Figure Lengend Snippet: Deletion of MAL-D and CCG-1423-derived inhibitors reduce hsp gene induction upon HS. Related to . A. Validation of MAL mutant flies. Expression of MAL-D and Act5C mRNAs in heterozygous (mal-dΔ7/+) and homozygous (mal-dΔ7) MAL-D mutant fly ovaries. Data is from three biological replicates and normalized to mal-dΔ7/+ values. The bar indicates the mean, individual data points are shown and error bars are standard errors of the mean (s.e.m). Statistical significances with student’s one-way t-test (*; P< 0.05). P-values: Mal-dΔ7/+ vs Mal-dΔ7 for Mal-d is 8E-7, Mal-dΔ7/+ vs Mal-dΔ7 for Act5c is 0.09. B. Knockdown efficiency of double stranded RNAs (dsRNAs) targeting different regions of MAL-D. Expression levels of MAL-D mRNAs in S2R+ fly cells treated with non-targeting dsRNA (dsGFP) and dsRNAs targeting MAL-D (dsMal-1-4). Data is from four biological replicates and normalized to dsGFP values, and shown as in A. Statistical significance with student’s one-sample t-test (*; P< 0.0125 with Bonferroni correction). P-values: dsMal-1 vs dsGFP is 0.002, dsMal-2 vs dsGFP is 0.002, dsMal-3 vs dsGFP is 0.016 and dsMal-4 vs sGFP is 0.039. C. Effect of MAL-D dsRNAs on Hsp70Aa mRNA induction. Fold changes in Hsp70Aa induction upon heat shock in S2R+ fly cells treated with MAL-D dsRNAs as in B. Data is from four biological replicates,and shown as in A. Statistical significance with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.0125 with Bonferroni correction). P-values: dsMal-1 vs dsGFP is 0.99, dsMal-2 vs dsGFP is 0.89, dsMal-3 vs dsGFP is 0.99 and dsMal-4 vs dsGFP is 0.99. D. Effect of 16h incubation with CCG-derived compounds on Act5C expression. Expression levels of Act5C mRNA in S2R+ cells treated with DMSO, CCG-1423 and CCG-203971 for 16 h. Data is from four biological replicates, normalized to DMSO values and shown as in A. Statistical significance with one-sample student’s t-test (*; P< 0.025 with Bonferroni correction). P-values: CCG-1423 vs DMSO is 0.006 and CCG-203971 vs DMSO is 0.024. E. Effect of 16h incubation with CCG-derived compounds on baseline expression of hsp genes. Expression level of Hsp68 and Hsp70Aa mRNAs in S2R+ cells treated with DMSO, CCG-1423 and CCG-203971 for 16 h. Data is from four biological replicates, normalized to the DMSO values and shows as in A. Statistical significance with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: CCG-1423 vs DMSO for Hsp68 is 0.055, CCG-203971 vs DMSO for Hsp68 is 0.39, CCG-1423 vs DMSO for Hsp70Aa is 0.06 and CCG-203971 vs DMSO for Hsp70Aa is 0.19. F. Effect of 16h incubation with CCG-derived compounds on heat shock induction of hsp genes. Fold change in Hsp68 and Hsp70Aa mRNAs upon 30 min heat shock in S2R+ cells treated with DMSO, CCG-1423 and CCG-203971 for 16 h. Data is from four biological replicates and shown as in A. Statistical significance with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: CCG-1423 vs DMSO for Hsp68 is 3E-8, CCG-203971 vs DMSO for Hsp68 is 0.047, CCG-1423 vs DMSO for Hsp70Aa is 8E-8 and CCG-203971 vs DMSO is 0.013. G. Effect of 30 min incubation with CCG-derived compounds on Act5C expression. Expression level of Act5C in S2R+ cells treated with DMSO, CCG-1423 and CCG-203971 for 30 min. Data is from five biological replicates, normalized to DMSO values and shown as in A. Statistical significance with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: CCG-1423 vs DMSO is 0.212, CCG-203971 vs DMSO is 0.468.

Article Snippet: CCG-1423 (sc-205241A; Santa Cruz Biotechnology) and CCG-203971 (SML1422; Sigma Aldrich) were dissolved to an initial concentration of 10 mM in DMSO.

Techniques: Derivative Assay, Mutagenesis, Expressing, Comparison, Incubation

Journal: bioRxiv

Article Title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

doi: 10.1101/2023.09.08.556810

Figure Lengend Snippet: Deletion of MAL-D and CCG-1423-derived inhibitors reduce hsp gene induction upon HS. A. Deletion of MAL-D reduces transcriptional induction of Hsp68 and Hsp70Aa genes in fly ovaries upon 20 min of heat shock at 37°C. Data is shown as fold changes in Hsp68 and Hsp70Aa mRNA induction in MAL-D-deleted (mal-dΔ7) and heterozygous (mal-dΔ7/+) flies. Data is from three biological replicates, with bar indicating the mean, individual data points shown and error bars are standard error of the mean (s.e.m). Statistical significance with student’s t-test (*; P< 0.05). P-values: Mal-dΔ7/+ vs Mal-dΔ7 for Hsp68 is 0.0249, Mal-dΔ7/+ vs Mal-dΔ7 for Hsp70Aa is 0.0054. B. Schematic representation of the experimental design in S2R+ cells for data shown in C and D. C. Effect of 30 min treatment with CCG-1423-derived inhibitors on baseline expression (non-heat shock; NH) of Hsp68 and Hsp70Aa . Data is from five biological replicates, normalized to DMSO treated sample and shown as in A. Statistical significance with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: CCG-1423 vs DMSO for Hsp68 is 0.066, CCG-203971 vs DMSO for Hsp68 is 0.064, CCG-1423 vs DMSO for Hsp70Aa is 0.096 and CCG-203971 vs DMSO is 0.108. D. CCG-1423 strongly inhibits HS-induced activation of Hsp68 and Hsp70Aa expression. Data is shown as fold change in mRNA induction, from five biological replicates and shown as in A. Statistical significance with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: CCG-1423 vs DMSO for Hsp68 is 8E-8, CCG-203971 vs DMSO for Hsp68 is 0.005, CCG-1423 vs DMSO for Hsp70Aa is 5E-8 and CCG-203971 vs DMSO is 0.032. See also for additional data.

Article Snippet: CCG-1423 (sc-205241A; Santa Cruz Biotechnology) and CCG-203971 (SML1422; Sigma Aldrich) were dissolved to an initial concentration of 10 mM in DMSO.

Techniques: Derivative Assay, Expressing, Activation Assay

Journal: bioRxiv

Article Title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

doi: 10.1101/2023.09.08.556810

Figure Lengend Snippet: MRTFs are dispensable for hsp induction in mammalian cells, but CCG-derived compounds inhibit hsp induction also in the absence of MRTFs A. Normalized [Reads Per Kilobase per Million mapped reads (RPKM)] coverage of MRTF-A and RNA polymerase II phosphorylated on serine 5 (Pol2S5P) on heat shock responding gene Hsph1 in mouse NIH 3T3 cells in non-heat shock (NHS) and heat shock (HS) conditions from ChIP-seq. B. Expression levels of hsp genes in the absence of MRTFs. Baseline expression of Hsph1 and Hspa1b mRNAs in wild type (MRTF_WT) and two MRTF-KO (MRTF_KO-1 and MRTF_KO-2) mouse NIH 3T3 cell lines. Data is from three biological replicates, normalized to WT values, bars represent the mean value, individual data points are shown and error bars are standard errors of the mean (s.e.m). Statistical significances are measured with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: MRTF_WT vs MRTF_KO-1 for Hsph1 is 0.08 MRTF_WT vs MRTF_KO-2 for Hsph1 is 0.043. MRTF_WT vs MRTF_KO-1 for Hspa1b is 0.14, MRTF_WT vs MRTF_KO-2 for Hspa1b is 0.13. C. Induction of hsp gene transcription in the absence of MRTFs. Fold change in transcriptional induction of hsp genes upon 20 min heat shock. Data is from three individual experiments and shown as in B. Statistical significances are measured with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.05). P-values: MRTF_WT vs MRTF_KO-1 for Hsph1 is 0.09, MRTF_WT vs MRTF_KO-2 for Hsph1 is 0.004, MRTF_WT vs MRTF_KO-1 for Hspa1b is 0.27 and MRTF_WT vs MRTF_KO-2 for Hspa1b is 0.13. D. Schematic representation of the experimental design in mouse NIH 3T3 cells for data shown in E. E. Effect of CCG-derived inhibitors on hsp gene induction in the presence and absence of MRTFs. Data is the fold change in Hsph1 mRNA induction upon 20 min of heat shock, from three individual experiments and shown as in B. Statistical significances are measured with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: CCG-1423 vs DMSO in MRTF_WT is 0.013, CCG-203971 vs DMSO in MRTF_WT is 0.0013, CCG-1423 vs DMSO in MRTF_KO-2 is 0.113 and CCG-203971 vs DMSO in MRTF_KO-2 is 0.0011. See also for additional data.

Article Snippet: CCG-1423 (sc-205241A; Santa Cruz Biotechnology) and CCG-203971 (SML1422; Sigma Aldrich) were dissolved to an initial concentration of 10 mM in DMSO.

Techniques: Derivative Assay, ChIP-sequencing, Expressing, Comparison

Journal: bioRxiv

Article Title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

doi: 10.1101/2023.09.08.556810

Figure Lengend Snippet: The effect of CCG-derived inhibitors is not limited to SRF-reporter genes. Related to . A. SRF reporter assay with serum response element (SRE)-controlled firefly luciferase activity normalized to constitutive renilla luciferase activity in cells treated with DMSO, CCG-1423 and CCG-203971 in serum starved conditions. Data is from three biological replicates, bars represent the mean, normalized to the mean of the DMSO values, individual data points indicated and error bars are s.e.m. Statistical significance with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.05). P-value: CCG-1423 vs DMSO is 0,92, CCG-203971 vs DMSO is 0.012. B. Normalized SRF reporter activity in serum-stimulated conditions shown as in A. Statistical significance with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.05). P-values: CCG-1423 vs DMSO is 0.007, CCG-203971 vs DMSO is 1E-00. C. SRE-controlled firefly luciferase activities in serum starved condition shown as in A. Statistical significance with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.05). P-values: CCG-1423 vs DMSO is 1E-00, CCG-203971 vs DMSO is 1E-00. D. SRE-controlled firefly luciferase activities in serum stimulated conditions shown as in A. Statistical significance with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.05).P-values: CCG-1423 vs DMSO is 1E-00, CCG-203971 vs DMSO is 6E-6. E. Renilla luciferase activities in serum starved condition shown as in A. Statistical significance with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.05). P-values: CCG-1423 vs DMSO is 1E-00, CCG-203971 vs DMSO is 1E-00. F. Renilla luciferase activities in serum stimulated condition shown as in A. Statistical significance with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.05). P-values: CCG-1423 vs DMSO is 1E-00, CCG-203971 vs DMSO is 1E-7.

Article Snippet: CCG-1423 (sc-205241A; Santa Cruz Biotechnology) and CCG-203971 (SML1422; Sigma Aldrich) were dissolved to an initial concentration of 10 mM in DMSO.

Techniques: Derivative Assay, Reporter Assay, Luciferase, Activity Assay, Comparison

Journal: bioRxiv

Article Title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

doi: 10.1101/2023.09.08.556810

Figure Lengend Snippet: CCG-1423-derived inhibitors act as general transcription inhibitors A. Effect of CCG-1423 and Actinomycin D (ActD) on baseline expression of Ecdysone responsive gene Eip74ef in S2R+ fly cells. Data is normalized to DMSO treated sample, from four biological replicates, bars represent the mean value, individual data points are shown and error bars are standard errors of the mean (s.e.m). Statistical significances are measured with student’s one-sample t-test (*; P< 0.025 with Bonferroni correction). P-values: DMSO vs ActD is 0.09 and DMSO vs CCG-1423 is 0.92. B. Effect of CCG-1423 and Actinomycin D (ActD) on transcriptional induction of Eip74ef upon 1 h of 20H-ecdysone stimulation. Data is fold changes in Eip74ef mRNA levels, from four biological replicates and shown as in A. Statistical significances are measured with one-way ANOVA followed by Tukey’s multiple comparison test (*; P< 0.025 with Bonferroni correction). P-values: DMSO vs ActD is 0.0006 and DMSO vs CCG-1423 is 0.021. C. Effect of CCG-derived compounds and ActD on nascent RNA production as measured by 5 ethynyl uridine (5-EU) incorporation in fly S2R+ cells. Top: Fluorescence microscopy images of representative cells, scale bar 30 microns. Bottom: Quantification of mean intensities of 5-EU staining in the indicated conditions from three biological replicates is shown as a 25th and 75th percentiles box plot, where the midpoint indicates the median value of EU intensities, the box represents mean and whiskers represent data range within 1.5IQR. Measurements from single cells shown as data points, with the total number of quantified cells indicated below the graph. Statistical significance with Mann-Whitney U-test (*; P< 0.017 with Bonferroni correction). P-values: DMSO vs ActD is < 0.0001, DMSO vs CCG-1423 is <0.0001, DMSO vs CCG-203971 is <0.0001. D. Effect of CCG-derived compounds and ActD on nascent RNA production as measured by 5 ethynyl uridine (5-EU) incorporation in mouse NIH 3T3 cells. Data is shown as in C. Statistical significance with Mann-Whitney U-test (*; P< 0.017 with Bonferroni correction). P-values: DMSO vs ActD is <0.0001, DMSO vs CCG-1423 is <0.0001, DMSO vs CCG-203971 is <0.0001.

Article Snippet: CCG-1423 (sc-205241A; Santa Cruz Biotechnology) and CCG-203971 (SML1422; Sigma Aldrich) were dissolved to an initial concentration of 10 mM in DMSO.

Techniques: Derivative Assay, Expressing, Comparison, Fluorescence, Microscopy, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

doi: 10.1101/2023.09.08.556810

Figure Lengend Snippet: Binding of RNA polymerase II to hsp genes in CCG-203971 treated mouse fibroblasts. Related to . A. Normalized (RPKM) coverage of RNA polymerase II phosphorylated on serine 5 (Pol2S5P) on heat shock responding gene Hsph1 in NIH 3T3 mouse cells in DMSO and CCG-203971 treated cells in non-heat shock (NHS) and heat shock (HS) conditions from ChIP-seq. B. Quantification of Pol2S5P on the gene body of the 15 hsp genes as normalized read counts (RPKM) in DMSO and CCG-203971 treated cells in non-heat shock (NHS) and heat shocked (HS) conditions in two replicate experiments. Data is shown as a 25th and 75th percentiles box plot, where the horizontal line represents the median, small box represents mean and whiskers represent data range within 1.5IQR. Statistical significance with Mann-Whitney U test (*; P< 0.05). C. Quantification of Pol2S5P around the transcription start site (TSS) of the hsp genes shown as in B. D. Quantification of Pol2S5P downstream of the CPS of the hsp genes shown as in B.

Article Snippet: CCG-1423 (sc-205241A; Santa Cruz Biotechnology) and CCG-203971 (SML1422; Sigma Aldrich) were dissolved to an initial concentration of 10 mM in DMSO.

Techniques: Binding Assay, ChIP-sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

doi: 10.1101/2023.09.08.556810

Figure Lengend Snippet: CCG-203971 results in accumulation of RNA polymerase II on the transcription start site in mouse fibroblasts A. Normalized (RPKM) coverage of RNA polymerase II phosphorylated on serine 5 (Pol2S5P) on the Rps26 gene in NIH 3T3 mouse cells in DMSO and CCG-203971 treated cells in non-heat shock (NHS) and heat shock (HS) conditions from ChIP-seq. B. Quantification of Pol2S5P on the gene body of the 11468 expressed genes in NIH3T3 mouse cells in DMSO and CCG-203971 treated cells in non-heat shock (NHS) and heat shock (HS) conditions from two replicate experiments. Data is shown as a 25th and 75th percentiles box plot, where the horizontal line represents the median, small box represents mean and whiskers represent data range within 1.5IQR. Statistical significance was calculated by Mann-Whitney U test. C. Quantification of Pol2S5P around the transcription start site (TSS) of the expressed genes shown as in B. Statistical significance was calculated by Mann-Whitney U test. (*; P< 0.05). P-values for NHS DMSO vs CCG-1423 in replicate 1 is 0.0183 and in replicate 2 is 3,9E-256. D. Quantification of Pol2S5P downstream of transcription end site (CPS) of the expressed genes shown as in B. Statistical significance was calculated by Mann-Whitney U test. (*; P< 0.05). P-values for NHS DMSO vs CCG-1423: replicate 1 is 0.000001.

Article Snippet: CCG-1423 (sc-205241A; Santa Cruz Biotechnology) and CCG-203971 (SML1422; Sigma Aldrich) were dissolved to an initial concentration of 10 mM in DMSO.

Techniques: ChIP-sequencing, MANN-WHITNEY