Journal: PLoS ONE

Article Title: Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer

doi: 10.1371/journal.pone.0230670

Figure Lengend Snippet: A) Identification of PC4 inhibitor AG-1031. An in vitro screening assay was developed based on the dsDNA binding activity of PC4. Reactions (in triplicate) were repeated twice with independent dilutions from the stock solutions of 34 active pharmaceutical ingredients (APIs) for non-oncology indications. The control reaction (Cont) contains no compound and represents the binding activity of PC4 to the dsDNA, which is normalized as the value “1”. AG-1031 (#31, highlighted) was identified as the most effective PC4 inhibitor. B) Interference of AG-1031 on PC4 binding to ds- or ssDNA. AG-1031 (#31) was identified as the most effective PC4 inhibitor by using an in vitro screen assay based on the dsDNA binding activity of PC4. The 50% inhibitory concentration (IC 50 ) of AG-1031 for dsDNA binding was 2.5 μM, and 15 μM for ssDNA binding, suggesting that AG-1031 preferentially inhibited binding of PC4 to dsDNA rather than to ssDNA. C) AG-1031 inhibits PC4 binding to monoclonal antibody and activator VP16. Recombinant PC4 was immobilized on a 96-well plate and incubated with either the monoclonal antibody against PC4 (mABG10) or recombinant 6His-tagged VP16 protein in the presence (#31) or absence (No inhibitor) of AG-1031. Bound monoclonal antibody was detected with anti-mouse HRP, and 6His-tagged VP16 was detected with anti-6His mouse mAb followed by anti-mouse HRP.

Article Snippet: The tumor slides were permeabilized with 0.3% Triton X100 in PBS for 5 min. After blocking non-specific staining by incubating the tissue for 30 min at room temperature in 3% bovine serum albumin, the tumor tissue slides were stained either with mouse monoclonal anti- PC4 antibody (AscentGene, USA, 1:1000 dilution) or with rabbit polyclonal anti-VEGFC (Vascular Endothelial Growth Factor C) antibody (Abcam, Cat# ab135506, USA, 1:500 dilution) at 4°C overnight.

Techniques: In Vitro, Screening Assay, Binding Assay, Activity Assay, Control, Concentration Assay, Recombinant, Incubation

Journal: PLoS ONE

Article Title: Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer

doi: 10.1371/journal.pone.0230670

Figure Lengend Snippet: In vitro transcriptions contained affinity purified RNA polymerase II, TFIID, and TFIIH and recombinant TFIIA, TFIIB, TFIIE, TFIIF, and the HNF-4 activator in the presence (A) or absence (B) of recombinant PC4. Drug additions were as indicated. Arrows identify the full-length G- free product originating at the authentic transcription start site. The asterisk in panel B identifies a non- specific RNA that is observed in the absence of PC4. Specific products were quantified and percent inhibition was plotted as a function of AG-1031 concentration (lower panels).

Article Snippet: The tumor slides were permeabilized with 0.3% Triton X100 in PBS for 5 min. After blocking non-specific staining by incubating the tissue for 30 min at room temperature in 3% bovine serum albumin, the tumor tissue slides were stained either with mouse monoclonal anti- PC4 antibody (AscentGene, USA, 1:1000 dilution) or with rabbit polyclonal anti-VEGFC (Vascular Endothelial Growth Factor C) antibody (Abcam, Cat# ab135506, USA, 1:500 dilution) at 4°C overnight.

Techniques: In Vitro, Affinity Purification, Recombinant, Inhibition, Concentration Assay

Journal: PLoS ONE

Article Title: Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer

doi: 10.1371/journal.pone.0230670

Figure Lengend Snippet: NSCLC cell lines A549, H1299, H841 and H460 were incubated in the presence of increasing concentrations of AG-1031. Viable cells were counted 48 hrs post-treatment by using a cell counting kit and measured using a 96- well plate reader at OD 450nm . AG-1031 showed inhibitory effect on all of four cell lines with IC50 between 1–2 μM (Fig 3A and 3B). Western blot analysis showed that treatment of A549 cells with AG-1031 resulted in down-regulation of PC4 in a concentration-dependent manner (Fig 3C/left panel). Expression of β-actin was used as the control. The density of PC4 expression was semi-quantified and the relative inhibition was plotted (Fig 3C/right panel).

Article Snippet: The tumor slides were permeabilized with 0.3% Triton X100 in PBS for 5 min. After blocking non-specific staining by incubating the tissue for 30 min at room temperature in 3% bovine serum albumin, the tumor tissue slides were stained either with mouse monoclonal anti- PC4 antibody (AscentGene, USA, 1:1000 dilution) or with rabbit polyclonal anti-VEGFC (Vascular Endothelial Growth Factor C) antibody (Abcam, Cat# ab135506, USA, 1:500 dilution) at 4°C overnight.

Techniques: Incubation, Cell Counting, Western Blot, Concentration Assay, Expressing, Control, Inhibition

Journal: PLoS ONE

Article Title: Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer

doi: 10.1371/journal.pone.0230670

Figure Lengend Snippet: A549 cell xenografts were produced in 7 mice and treated with AG-1031 (“AG-1031”) every other day. The tumor size was measured and compared to the control group of 6 mice without AG-1031 treatment (“vehicle”). Panel A compares the average tumor size between treated (“AG-1031”) and untreated (“vehicle”) groups. Western blot analysis revealed a decreased level of PC4 protein in most AG-1031-treated tumor tissues compared to untreated tumor tissues (panel B). Expression of TATA binding protein (TBP) was used as an internal control. The density of PC4 expression was measured and the relative inhibition was plotted (Fig 4C).

Article Snippet: The tumor slides were permeabilized with 0.3% Triton X100 in PBS for 5 min. After blocking non-specific staining by incubating the tissue for 30 min at room temperature in 3% bovine serum albumin, the tumor tissue slides were stained either with mouse monoclonal anti- PC4 antibody (AscentGene, USA, 1:1000 dilution) or with rabbit polyclonal anti-VEGFC (Vascular Endothelial Growth Factor C) antibody (Abcam, Cat# ab135506, USA, 1:500 dilution) at 4°C overnight.

Techniques: Produced, Control, Western Blot, Expressing, Binding Assay, Inhibition

Journal: PLoS ONE

Article Title: Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer

doi: 10.1371/journal.pone.0230670

Figure Lengend Snippet: Tumor tissues from AG-1031-treated A549 xenograft (#757) and from untreated xenograft (#753) were stained either with monoclonal antibody against PC4 (A and B) or with polyclonal antibody against VEGFC (C and D). Primary antibodies were detected using IgG-conjugated with rhodamine (A and B), Alexa Fluor 568 (C and D) and nuclei with DAPI (A, B, C and D).

Article Snippet: The tumor slides were permeabilized with 0.3% Triton X100 in PBS for 5 min. After blocking non-specific staining by incubating the tissue for 30 min at room temperature in 3% bovine serum albumin, the tumor tissue slides were stained either with mouse monoclonal anti- PC4 antibody (AscentGene, USA, 1:1000 dilution) or with rabbit polyclonal anti-VEGFC (Vascular Endothelial Growth Factor C) antibody (Abcam, Cat# ab135506, USA, 1:500 dilution) at 4°C overnight.

Techniques: Staining