Journal: Cell reports

Article Title: Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation.

doi: 10.1016/j.celrep.2016.04.066

Figure Lengend Snippet: Figure 1. PD407824 Sensitizes C2C12 Myoblasts to BMP4-Induced Activation of BMP Signaling (A) Scheme of the high-throughput screening. (B) Chemical structure of PD407824. (C) Dose curve of PD407824 on C2C12 myoblasts transfected with pId2-Luc-GFP reporter. (D) PD407824 sensitizes C2C12 myoblasts to BMP4-induced upregulation of endogenous Id2 expression measured by qRT-PCR. (E) PD407824 sensitizes C2C12 myoblasts to BMP4-induced SMAD1/5/9 phosphorylation indicated by western blotting. (F) Quantification of relative SMAD1/5/9 activity. The phosphorylation levels were normalized to total SMAD1 protein. (G) PD407824 sensitizes C2C12 myoblasts to BMP4-induced upregulation of Id1 expression measured by qRT-PCR. See also Figure S1.

Article Snippet: Cell Culture C2C12 myoblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, High Throughput Screening Assay, Transfection, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Activity Assay

Journal: Cell reports

Article Title: Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation.

doi: 10.1016/j.celrep.2016.04.066

Figure Lengend Snippet: Figure 2. PD407824 Synergizes with BMP4 to Reprogram C2C12 Myoblasts into Mature Osteoblasts (A) C2C12 myoblasts were reprogrammed into cells expressing osteogenic marker alkaline phosphatase (ALK). (B) Quantification of ALK activity by fluorescence-based substrate. (C) qRT-PCR to detect the expression of mature osteoblast markers, including collagen I (collagen), osteopontin (OPN), and osteocalcin (OC). (D) Alizarin red S staining of the mature osteoblasts. PD: PD407824. Scale bar, 50 mm.

Article Snippet: Cell Culture C2C12 myoblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Marker, Activity Assay, Quantitative RT-PCR, Staining

Journal: Cell reports

Article Title: Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation.

doi: 10.1016/j.celrep.2016.04.066

Figure Lengend Snippet: Figure 5. PD407824 Functions through Inhibiting CHK1 (A and B) CHIR124, a CHK1 inhibitor, mimics the effect of PD407824 to sensitize BMP4-induced upregulation of Id2 expression (A) and increased SMAD1/5/9 phosphorylation (B) in C2C12 myoblasts. The relative Id2 transcript level was normalized to DMSO treatment conditions. The relative SMAD1/5/9 phosphorylation was normal- ized to total SMAD1. (C) Relative Id2 transcript expression in CHK1+/

Article Snippet: Cell Culture C2C12 myoblasts were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Phospho-proteomics

Journal: Cancers

Article Title: Advances in Prognostic Methylation Biomarkers for Prostate Cancer

doi: 10.3390/cancers12102993

Figure Lengend Snippet: Methylation profiling approaches for biomarker discovery and validation. ( A ) Candidate gene approaches: Targeted candidate gene approaches used in a priori studies and for the validation of novel candidate markers. ( B ) Genome wide approaches: Microarray and sequencing-based genome-wide approaches used for the discovery of novel biomarkers—comparison of the methodology, number of CpGs (cytosine-guanine dinucleotides) and/or genes targeted and coverage of the methylome across the different platforms. RefSeq: Reference Sequences; PCR: polymerase chain reaction; MSP: methylation-specific PCR; ddPCR: droplet digital PCR; COBRA: combined bisulphite restriction analysis; qMSP: quantitative methylation-specific PCR; HM: human methylation; RRBS: reduced representation bisulphite sequencing; MBDCap-Seq: methyl-CpG binding domain capture sequencing; WGBS: whole genome bisulphite sequencing.

Article Snippet: These studies used targeted methylation profiling techniques including methylation-specific PCR (MSP) [ ], quantitative methylation-specific PCR (qMSP) [ ], pyrosequencing [ , ] and mass spectrometry (MassARRAY EpiTYPER, Agena Bioscience, San Diego, California, USA) [ ] to assess the methylation profile of a specific gene of interest ( ). lists other targeted approaches that have also been used to assess methylation in cancer, including ddPCR [ ], COBRA [ ], high resolution melt curve [ ] and headloop MSP [ , , ].

Techniques: Methylation, Biomarker Discovery, Genome Wide, Microarray, Sequencing, Comparison, Polymerase Chain Reaction, Digital PCR, Combined Bisulfite Restriction Analysis Assay, Bisulfite Sequencing, Binding Assay

Journal: Frontiers in Immunology

Article Title: STING activation by teniposide: a potential direct mechanism beyond cGAS stimulation

doi: 10.3389/fimmu.2025.1677836

Figure Lengend Snippet: Teniposide is a potential STING agonist that induces IFNB1 gene expression. (a) Schematic representation of high-throughput virtual screening HTVS of NIH libraries targeting the STING binding pocket. (b) Representation of the 2D structures of Teniposide and cGAMP. (c) Relative IFNB1 gene expression in THP1 cells treated with varying doses of Teniposide or cGAMP. (d) Time-course IFNB1 gene expression in THP1 cells treatment with cGAMP or Teniposide. (e) MX1 and IL6 gene expression in THP1 cells 12h post-Teniposide treatment. (f) Dose response of Teniposide in BMDMs, showing relative IFNB1 expression 24h post-treatment. (g) IFNB1 time-course expression in BMDM upon stimulation with Teniposide (1µM or 3µM). cGAMP induction at 8h was used as a reference control. (h) Relative MX1 and IL6 gene expression in BMDM 48h after Teniposide treatment. Statistical significance is indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.005, **** p ≤ 0.001.

Article Snippet: Thp1 ATCC ® TIB-202TM cell cultures are human monocyte cells obtained from peripheral blood of patients with acute monocytic leukemia.

Techniques: Gene Expression, High Throughput Screening Assay, Binding Assay, Expressing, Control

Journal: Frontiers in Immunology

Article Title: STING activation by teniposide: a potential direct mechanism beyond cGAS stimulation

doi: 10.3389/fimmu.2025.1677836

Figure Lengend Snippet: Teniposide-induced IFNB1 expression requires STING and may occur independently of cGAS and IFI16. (a) IFNB1 relative gene induction measured by RT-qPCR in WT or STING KO THP1 cells after Teniposide 3µM treatment for 8h. (b) IFNB1 relative expression in WT or GT STING BMDM treated with 3µM Teniposide for 8h. (c) Western blot representing pTBK1, total TBK1, pIRF3, total IRF3 and bacting in THP1cells mock treated, treated for 4h with 3µM of Teniposide or 1µM of diABZI. (d) IFNB1 relative gene expression in WT THP1 cells stimulated for 18h with 10 IU of IFN-β/ml or mock-treated followed by a treatment with DMSO, cGAMP or Teniposide (3µM). (e) Same as in (d) but in WT and cGAS KO THP1 cells. (f) Same as in (d) but in WT and IFI16 THP1 KO cells. (g) Western blot verifying STING, cGAS or IFI16 in WT but not STING, cGAS, and IFI16 KO THP1 cells. Tubulin or β-Actin is shown as a loading control. Statistical significance is indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.005, **** p ≤ 0.001.

Article Snippet: Thp1 ATCC ® TIB-202TM cell cultures are human monocyte cells obtained from peripheral blood of patients with acute monocytic leukemia.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Gene Expression, Control

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Diagram of growth competition assay. mCherry-labelled RPE1 cells were mixed with unlabeled TP53 −/− RPE1 (1:1), exposed to IR and grown for 6 days. b, Relative abundance of unlabeled TP53 −/− Clone#1 measured by Intellicyte high-throughput cytometry ± SEM (n=6) is shown, normalized to the untreated (0Gy) cohort at each time point. c, Relative abundance of unlabeled TP53 −/− Clone#2 ± SEM (n=6) is shown, normalized to the untreated (0Gy) cohort at each time point. d, Representative immunofluorescence images of 53BP1 foci in cells with indicated genotypes untreated (no IR) or treated with IR (5Gy) and collected at .5, 2, and 4 h after irradiation. e, Quantification of 53BP1 foci. Data shown are mean (n=50 cells per treatment condition) ± SEM (n=3), and are consistent across two independent biological replicates. * p <0.05; ** p <0.01; by two-tailed Student’s t-test. f, Representative Neutral COMET fluorescence staining for DNA tails in cells with indicated genotypes treated with or without 5Gy IR. For irradiated cells, 2 timepoints are shown: immediately after and 4 hours post IR. COMET tails and heads are denoted by OpenComet software analysis. g, Quantification of DNA DSBs via Neutral COMET assay reported as tail DNA percent at 0 and 4 hours post IR in RPE1 and two TP53 −/− RPE1 cell lines. Data shown are mean (n= 50-150 cells per treatment condition) ± SEM, and are consistent across three independent biological replicates. * p <0.05; ** p <0.01; **** p <0.0001 by two-tailed Student’s t-test.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Competitive Binding Assay, High Throughput Screening Assay, Cytometry, Immunofluorescence, Irradiation, Two Tailed Test, Fluorescence, Staining, Software, Neutral Comet Assay

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Live cell imaging procedure. Cells transfected with 10 nM si-control or si- TP53 for 48 h prior to imaging. 18 h into imaging, cells are treated with NCS (100 nM), DNA-PKi (.5 uM) or both and imaged for 72 total hours. b, RPE1 cell expressing the PCNA-mCherry and 53BP1-mVenus reporters. Cell cycle phases delineated by PCNA foci and DNA DSBs are marked by 53BP1 foci. c, RT-qPCR for TP53 mRNA levels (left) and CDKN1A mRNA levels (right) in si-control treated vs. si- TP53 treated cells. To induce CDKN1A expression, cells irradiated at 5Gy and mRNA harvested 3 hrs post IR. d, Heatmap of 53BP1 foci tracings for single cells tracked from birth to mitosis or end of imaging. For si-control (n = 30 cells) and si- TP53 treated RPE1 (n = 60 cells) treated with NCS 100 ng/ml. For visualization, cells are aligned to 10 frames prior to drug addition (black arrow). e, Heatmap of 53BP1 foci tracings for si-control (n = 25 cells) and si- TP53 treated RPE1 cells (n = 55 cells) treated with 100 ng/ml NCS + 0.5 uM DNA-PKi. f, Peak 53BP1 foci counts for cells treated with 100 ng/ml NCS or NCS+0.5 uM DNA-PKi. Significance determined using two-tailed t-test. g, Area under the curve (AUC) analysis of 53BP1 burden showing integral DNA damage for cells treated with NCS vs. NCS and DNA-PKi. Cells are segregated into two groups: cells exposed to drug in G1 vs. S phase (n = 25-30 G1or S cells for si- TP53 cohort, n = 10-15 G1 or S cells for si-control cohort). Significance determined by two-tailed t-test. **** p <0.0001, *** p <0.001, n.s. = non-significant. h, 53BP1 foci burden in G1 vs. S phase p53-deficient RPE1 upon exposure to NCS and DNA-PKi. Dashed line = S phase onset, blue line = mean 53BP1 foci burden for all cells in G1 with NCS and DNA-PKi addition, orange line = mean foci value for cells in G1 with NCS treatment alone, (n = 30 cells for each condition).

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Live Cell Imaging, Transfection, Control, Imaging, Expressing, Quantitative RT-PCR, Irradiation, Two Tailed Test

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Normal Mitosis: RPE1 Cell cycle representative of normal mitosis, with NCS treatment only. For all cells in this figure both the PCNA and the 53BP1 channels are shown as two individual movies. b, Transient G2 Delay: RPE1 cell cycle representative of a transient cell cycle delay in G2 (length of G2 is significantly prolonged in comparison to untreated cells). This cell was treated with NCS and DNA-PKi. c, G1 Arrest: RPE1 cell cycle representative of a permanent G1 arrest. This is a p53 proficient cell treated with NCS and DNA-PKi.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Comparison

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Cell cycle outcome analyses for si-control treated RPE1, dashed white line indicates drug addition, each row is an individual cell (n = 60 cells for NCS and n=60 cells for NCS+DNA-PKi treatment). Colored bars indicate different phases of the cell cycle, legend shown with no treatment control for comparison. Cells with red bars at the end of mitosis indicate terminal cell cycle event (mitotic catastrophe or apoptosis). Event frequency is reported as a percentage on the right. Cells exposed in G1 vs. S cells are treated as separate cohorts. Fisher’s exact test was performed between −/+ DNA-PKi cohorts using 2 outcome groups (viable, vs. non-viable (arrested cells + terminal outcomes). **** p <0.0001, n.s. =non-significant b, Cell cycle outcome analyses for si- TP53 treated RPE1, dashed line indicates drug addition, each row is an individual cell (n = 60 cells for NCS and n=60 cells for NCS+DNA-PKi treatment). c, AUC analysis of 53BP1 damage burden in viable vs. non-viable p53-deficient cells that were treated with NCS and DNA-PKi. Statistical significance was calculated using a Mann-Whitney test comparing ranks. **** p <0.0001 d, Dynamics of 53BP1 foci burden p53-deficient RPE1 segregated by mitotic viability. The red line corresponds to mean 53BP1 foci burden for all p53-deficient cells treated with NCS and DNA-PKi that undergo catastrophic mitoses, black line indicates mean foci value for p53-deficient cells with NCS and DNA-PKi treatment that are viable post mitosis, (n = 20 viable cells and n = 33 non-viable cells).

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Time stamped image sequence of apoptotic cell (PCNA channel shown). Cells that experienced nuclear degradation during cell cycle prior to mitosis were categorized as “apoptotic cells.” In this sequence a cell in G2 experiences cell death at 27 hours post birth, with indication of mitotic attempt, with nuclear envelope collapse or presence of any daughter cells. b, Time stamped image sequence of cell that experienced mitotic catastrophe (PCNA channel shown). Cell undergoes nuclear envelope collapse (24:10), and attempts mitosis, in subsequent images fragmentation of nucleus is clearly visible with no viable daughter cells present. Cell non-viability during mitosis was defined as mitotic catastrophe. c, Integral DNA damage burden for p53-deficient cells treated with NCS (100 ng/ml) and DNA-PKi (.5 uM) are calculated and segregated by viable (black) vs. non-viable outcomes (red). Legend indicates which phase of cell cycle the cells are in during drug exposure, followed by the phase for which the burden is calculated. Ex: G1 cells G1 burden = cells in G1 during drug exposure and total damage burden in G1. Area under the curve (AUC) analysis was performed by plotting 53BP1 foci counts over time for each cell and integrating burden over time. Statistical significance was determined using two-tailed Student’s t-test.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Sequencing, Two Tailed Test