Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Cells were treated with increasing concentrations of cisplatin for 72 hr and % survival was measured using the Crystal Violet assay. LXSN, E6, E7, E6E7 expressing cells showed respective IC50 of 2.5, 0.74, 1.27 and 0.73 uM; 72 hr. (B-D) Cells were transfected with siControl or siFancD2 for 48hrs and plated for western blotting and cisplatin survival assay. (B) Western blot showing FancD2 knockdown in the cells harvested at the time of reading survival assay. (C) Survival curves of LXSN, E6, E7 and E6E7 cells transfected with siRNA (siFancD2 or siControl) and treated with indicated doses of cisplatin for 48 hr. (D) Survival curve of siControl cells treated with indicated doses of cisplatin for 48 hrs.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Crystal Violet Assay, Expressing, Transfection, Western Blot, Clonogenic Cell Survival Assay, Knockdown

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Immunoblot showing FancD2/ FancI expression and monoubiquitination status in transduced HFK cells which were either untreated or treated with 3 uM cisplatin for 24 hr. Ub refers to the monoubiquitinated forms of FancD2 and FancI, and non-Ub refers to the non-ubiquitinated forms. Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: Non-Ub) and total FancD2 (Ub + Non-Ub) levels are indicated beneath the corresponding lanes. (B) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells that were either untreated or treated with 3 uM cisplatin for 24 hr. Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (C) Immunoblot of whole cell lysates showing levels of phosphorylated S556 FancI, total FancI, Ub-PCNA, non-Ub PCNA, and UHFR1. Vinculin acts as a loading control.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Western Blot, Expressing, Control

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) HFK cells were treated with cisplatin (3 uM) for 24 hr and immunostained with FancD2 (red), pH2AX (green) and DAPI (blue). Representative images are shown. (B) Cells with >5 foci were counted, and the percentage of positive cells is plotted (n = 3, mean ± SEM). (C) Quantification of percentage of FancD2 foci co-localization with pH2AX with or without cisplatin treatment. * (p-value ≤ 0.05) and ** (p-value ≤ 0.01) denote a statistically significant difference from the similarly treated LXSN control cells. Error bars represent standard error of the mean. Quantification was based on data observed from ≥ 15 nuclei from three independent experiments. (D) U2OS-DR cells (transduced with LXSN or E6/E7) were transfected with I-SceI expression plasmid for 24 hr before fixation. Cells were immunostained with pH2AX, FancD2, and DAPI (blue). Cells with a single large pH2AX focus (red) were examined for the colocalization with FancD2 (green). Representative images are shown. (E) Quantification of the frequency of colocalization of FancD2 with pH2AX foci in U2OS-DR cells. Data represent mean ± SEM and was based on observations from ≥ 50 cells from at least three independent experiments. (F-H) U2OS-DR cells transduced with the indicated constructs were transfected with the siControl or siFancD2. They were transfected with I-SceI expression plasmid and then fixed after 24 hr of transfection and stained with Rad51 and pH2AX antibodies. (F) Cell lysates were subjected to western blotting to confirm depletion of FancD2. (G) Cells with a single large pH2AX focus (red) were inspected for the colocalization with Rad51 (green). Representative images are shown. (H) Quantification of the frequency of colocalization of Rad51 with pH2AX foci. Data represent mean ± SEM and was based on observations from ≥ 25 cells from at least three independent experiments. * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas n.s. indicates non-significant.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Control, Transduction, Transfection, Expressing, Plasmid Preparation, Construct, Staining, Western Blot

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Outline of an experiment to evaluate FancD2 monoubiquitination/de-ubiquitination pattern. Transduced HFK cells were untreated or treated with cisplatin (1.5 uM for 24 hr) or exposed to 10 mJ/cm 2 UVB and allowed to repair. Whole-cell lysates were prepared for immunoblot at various time points during the experiments (represented by Δ). (B-C) Immunoblots of HFKs subjected to the experiment outlined in , following cisplatin withdrawal (B) or recovery after UVB exposure (C). Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: non-Ub) are indicated beneath the corresponding lanes. FancD2 Ub:non-Ub ratio in cells following cisplatin withdrawal and UVB exposure was plotted alongside Figure 5B and 5C. ** (p< 0.01) denotes a statistically significant difference from 0 hr cisplatin withdrawal. ‘ns’ denotes non-significant differences. (D) USP1 immunoblot in cells untreated and treated with cisplatin. (E) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells subjected to the experiment outlined in . Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (F) Immunoblot showing levels of p-FancI-S565 and total FancI in cisplatin-treated or untreated cells. (G) Immunoblot for p-ATR, pCHK1, FancD2 and p-FancI-S565 following cisplatin withdrawal for 18 and 24 hrs. Actin or vinculin act as a loading control for immunoblots (B-G). (H) Proposed mechanisms for delayed de-ubiquitination of FancD2 in E6 cells.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Ubiquitin Proteomics, Western Blot, Control

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Outline of synchronization assay in HFK cells using double-thymidine block. Cells were synchronized by double-thymine block and released at various time points. Immunoblot for FancD2, FancI, cyclin A and vinculin of LXSN (B) and E6 cells (C). Cell-cycle phases at each time point were determined by flow cytometry of DNA content (D). Asynchronous (Asyn.) cells, which have not undergone thymidine block, were included for comparison.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Blocking Assay, Western Blot, Flow Cytometry, Comparison

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Immunoblot for p53 (upper panel) and RT-PCR analysis of 16E6 and GAPDH expression (lower panel) in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T). (B-C) Immunoblot showing FancD2 expression and monoubiquitination status in cells which were either untreated or treated with cisplatin (B) or nutlin (C) at 1.5 uM for 24 hr. (D-E) Cells were untreated or treated with cisplatin (E) or exposed to 10 mJ/cm 2 UVB (D) and processed similarly as in .

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transduction, Mutagenesis

Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Representative projection images of anti-Sall4 (white), anti-Oct4 (magenta) and DAPI (blue) staining in WT (n=3), Sall4 Δ/+ (Het, n=2) and Sall4 Δ/Δ (KO, n=3) 16 cell stage embryos. Scale bars = 50μM. (B)Representative projection images of DAPI (blue), anti Nanog (green), anti-Oct4 (magenta) and anti-Sall4 (white) staining in control (Het; N = 10) and Sall4 Δ/Δ (KO; N = 5) early blastocyst embryos (E3.5). Scale bars = 50μM. (C)Quantification of the number of cells per embryos from (B) (left), the percentage of Nanog positive cells per embryo (middle), and intensity values for the Nanog positive cells from (B) for control (Wt/Het) and KO embryos (right). Significance was determined using a Mann-Whitney test for all three comparisons (*** P≤ 0.001). (D)Representative projection images of anti-Nanog (green), anti-Gata4 (magenta), anti-Cdx2 (white) staining, plus DAPI (blue) in control (Het; n=7) and Sall4 Δ/Δ (KO, n=6) E3.75 embryos. Scale bar = 50μM. (E)Quantification of the number of cells per embryos from (D) (left), the percentage of Nanog positive cells per embryo (middle), and percentage of Gata4 positive cells per embryo (right) for control (Wt/Het) and KO embryos. Significance was determined using a Mann-Whitney test (** P≤ 0.01). (F)Single cell gene expression analysis from E3.25 ICM WT/Het and KO embryos at indicated stages, with Log 10 expression normalised to that of housekeeping genes (see Methods) on the y-axis. Each dot represents a single cell, and each plot shows data for the indicated gene. Statistical significance was calculated using a Kruskall-Wallis test (Supplemental Table 1), * indicates P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P <=0.0001. Published data from wild type 16-cell embryos are included for comparison . (G)Same as in (F), but for genes expressed in primitive endoderm cells.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Staining, MANN-WHITNEY, Expressing

Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Representative projection images of anti-Nanog (green), anti-Gata6 (magenta) staining, along with composite images of anti-Nanog and anti-Gata6 together, plus the inclusion of anti-Cdx2 (white) staining in control embryos (WT n=4) and Sall4 Δ/Δ (KO, n=5) E3.75 embryos. Scale bar = 50μM. (B)Representative projection images of anti-Nanog (green), anti-Gata4 (magenta), anti-Cdx2 (white) staining in control (WT n=9) and Sall4 Δ/Δ (KO, n=6) E4.5 late blastocysts. Scale bar = 50μM. (C)Quantification of the number of cells per embryo (left); the percentage of Nanog positive cells per embryo (middle); and the percentage of Gata4 positive cells per embryo at 4.5 dpc. *** P ≤0.001, **** P ≤0.0001 by Mann-Whitney test. (D)Representative confocal projection images of control (WT or Het) and Sall4 Δ/ Δ (KO) embryos flushed at 2.5dpc and cultured for 48hours in media only or with 0.5 μg/μl or 1 μg/μl Fgf2. Embryos were stained with anti-Gata4 (magenta), anti-Nanog (green) and anti-Cdx2 (white). Scale bar = 50μM. N=9 for WT or Het embryos cultured without Fgf2 and in 0.5μg/ml Fgf2, n=10 for those in 1μg/ml Fgf2. N=4 for KO embryos cultured without Fgf2 and in 1μg/ml and n=3 for those cultured in 0.5μg/ml Fgf2. (E)Percentage of Nanog positive (top) or Gata4 positive (middle) cells per embryo for control (WT and Het) or Sall4-null (KO) embryos after culture in indicated conditions for 48 hours. Each point represents one embryo. Statistical significance was tested using an unpaired Mann-Whitney test: * P ≤0.05, *** P ≤0.001.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Staining, MANN-WHITNEY, Cell Culture

Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Diffusion plot of single cell gene expression data for Sall4-null (KO, left) and wild type (WT, right) ICM cells. Points represent individual cells of the indicated stages and genotypes. 8- and 16-cell data is taken from . (B)Heat map constructed from single ICM cell expression data based on hierarchical clustering. Normalised Ct values refer to –ΔCt values normalised to housekeeping genes ( Ppia, Gapdh and Hprt ). Each row is one cell with the genotype and the time isolated indicated by the annotation panel on the left and key on the right (dpc = days post coitum). Each column shows expression of one gene. The cells cluster into 5 groups which have been labelled according to their expression profiles: Early unspecified (EU), Making a decision (MD), Epiblast (Epi), Primitive Endoderm (PrE) and the outliers. (C)The percentage of cells in each cluster from (B) at each time point (E3.25 or E3.75) for KO and control (WT, including cells from Het embryos) ICM cells. (D)Absolute average correlation (see Figure S3) between cells for each developmental stage is plotted for control (WT, black) and Sall4-null (KO, blue) cells. CV for 8- and 16-cell stages were calculated using data from O’Shaugnessy-Kirwan et al 2015. Error bars represent SEM, and significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test. *** P≤0.001, **** P≤0.0001.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Diffusion-based Assay, Expressing, Construct, Isolation

Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Single cell expression data for genes characteristic of Epiblast and Primitive Endoderm split into clusters as specified in for control (WT, red) and Sall4-null (KO, Blue) cells. Each dot represents a single cell. Statistical significance was calculated using a Kruskall-Wallis test (Supplemental Table 1): * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P <=0.0001. (B)Model of how Sall4 facilitates lineage commitment. In early wild type ICMs (top left) most cells (purple circles) exist in an attractor state of a differentiation landscape associated with an unspecified identity (EU), though some cells occupy the MD state, which is essentially a ‘staging post’ en route towards differentiation. The probability of achieving a differentiated state (PrE or Epi) is very low. As the embryo matures (top right) the depth of the attractor state lessens and cells in the MD state are able to commit to either the PrE or Epi state. In the absence of Sall4 (bottom panels) the depth of the EU attractor state is more pronounced, and cells within this state show increased cell-cell variability. As the embryo matures the depth of the attractor is lessened, but remains deeper than in wild type cells so most Sall4-null cells still occupy the MD state and few have committed to either PrE or Epi. Black horizontal lines represent the threshold limits of the EU and MD states.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Expressing

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a , Cryo-EM reconstruction of the left-handed filaments of ANXA11 and TDP-43 from FTLD-TDP Type C, shown parallel to the helical axis. b, Identification of TDP-43 and ANXA11 chains in the ordered filament fold. ANXA11 was identified by deriving a sequence motif directly from well-resolved amino acid side chain densities in the cryo-EM reconstruction (see Methods). c, Cryo-EM reconstruction and atomic model of the filaments, shown for single TDP-43 and ANXA11 chains perpendicular to the helical axis. The green arrow indicates an isolated peptide consistent with TDP-43 residues N352-G357. Buried ordered solvent is indicated with red dots. d and e, Domain organisation of TDP-43 (d) and ANXA11 (e). DIX, dishevelled and axin domain; N, nuclear localisation signal; RRM, RNA-recognition motif; LCD, low-complexity domain; ANX, annexin repeat. The black lines indicate the regions that form the filament fold. f and g, Amino acid sequence alignment of the secondary structure elements of the TDP-43 (f) and ANXA11 (g) chains. Arrows indicate -strands. The sequences that form the interface between TDP-43 and ANXA11 are underlined. a-c, Cryo-EM density for TDP-43 is in grey and ANXA11 is in yellow. b, c, f and g, The TDP-43 glycine-rich (G284-G310, magenta), hydrophobic (M311-S342, white) and QIN-rich (Q343-Q345, green) regions are highlighted. ANXA11 is shown in orange.

Article Snippet: For immuno-EM, filament extracts were deposited onto carbon-coated 300-mesh copper or nickel grids (Nissin EM and Electron Microscopy Sciences, respectively), blocked with 0.1 % gelatin in PBS, and incubated with primary antibodies against pS409/410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:50) and N-terminal Annexin A11 (Proteintech, 10479-2-AP, 1:50) in PBS containing 0.1% gelatin at 21 °C for 3 h or at 4°C overnight.

Techniques: Cryo-EM Sample Prep, Sequencing, Isolation, Solvent

Journal: BMC Biology

Article Title: DNA damage strength modulates a bimodal switch of p53 dynamics for cell-fate control

doi: 10.1186/1741-7007-11-73

Figure Lengend Snippet: Bimodal switch of p53 dynamics modulates the p53 cytoplasmic localization. (a) Representative single-cell trajectories of average p53 fluorescence in the nucleus (black line) and cytoplasm (red line) under 100 μmol/l etoposide. (b) Western blotting analysis of p53 levels and the indicated p53 post-translational modifications in the nucleus and cytoplasm of U-2 OS cells treated with no drug, 1 μmol/l etoposide (treated for 24 hours) and 100 μmol/l etoposide (treated for 12 hours), respectively. To confirm the quality of the subcellular fractionation, tubulin was used as a cytoplasmic marker and transcriptional factor II B (TFIIB) as a nuclear marker.

Article Snippet: Antibodies used were: PARP1 (#9542), p21 (#2947), Puma (#4976), TFIIB (#4169), phospho-p53 (Ser20) (#9287), phospho-p53 (Ser46) (#2521) and acetyl-p53 (Lys382) (all Cell Signaling Technology/(Danvers, MA, USA); acetyl-p53 (Lys120; #BAM-71-131-EX; Cosmo Bio, Yokohama, Japan); p53 (#sc-126), Mdm2 (#sc-965) and Bax (#sc-493) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-histone H2A.X (#06-570; Millipore Corp., Billerica, MA, USA).

Techniques: Fluorescence, Western Blot, Fractionation, Marker