Journal: Cell Reports Medicine

Article Title: Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities

doi: 10.1016/j.xcrm.2021.100240

Figure Lengend Snippet:

Article Snippet: JMS-cl.1 and JMS-cl.2 hiPSCs were transduced with lentiviruses encloding for the non-specific control short-hairpin RNA (shControl; pLKO.1 puro (Addgene ID: 8453)) or the p53 targeting shRNAs; shP53a (shp53 pLKO.1 puro shRNA (Addgene ID: 19119) ), shP53b (shp53 pLKO.1 puro shRNA-427 (Addgene ID: 25636) ) and shP53c (shp53 pLKO.1 puro shRNA-941(Addgene ID: 25637) ).

Techniques: Virus, Recombinant, Knock-Out, Library Quantification, RNA Sequencing, Control, Mutagenesis, shRNA, Software, Reverse Transcription, SYBR Green Assay, Imaging

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 2. p53 Restrains MYC-Induced Epidermal Differentiation (A) Expression of GFP (green) and p53 (red) by immunofluorescence of HKMYCER cells infected with control vector CTGFP (CT) or with shP53 as indicated after activation of MYC by addition of OHT for 3 days. Note that within the mixed cul- tures, p53 is undetected in cells expressing shP53 (green cells, broken line). (B) Detection of p53 and its target p21 by western blot in CT or shP53 cells treated with OHT for 3 days as indicated. Bar histograms represent quantitation of the western blots normalized to GAPDH (GDH). (C) Expression of p53 as measured by RT-PCR in CT or shP53 cells after activation of MYC by OHT for 3 days. (D) Typical phase contrast and green fluorescence (GFP) of live shP53 cells 4 days postinfection with or without OHT as indicated. (E) Percent of CT or shP53 GFP cells expressing the terminal differentiation marker keratin K1 after addition of OHT for 5 days, as determined by immunofluorescence. (F) Immunofluorescence for involucrin (green) of CT or shP53 cells, 5 days after addition of OHT. Nuclear DNA by DAPI (blue). (G) Percent of CT or shP53 cells expressing in- volucrin (Invol) after addition of OHT for 5 days, as determined by flow-cytometry. Error bars are SEM of duplicate samples of representative experiments. *p < 0.05. Scale bar, 50 mm.

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Activation Assay, Western Blot, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Cytometry

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 3. Disruption of Endogenous p53 in Human Keratinocytes Causes Replication Stress (A) Expression of p53, its target p21, and p63 in HK infected with CTGFP (CT) or with shP53 as indicated, untreated or treated with the genotoxic drug doxorubicin (DOXO) for 24 hr. Bar histogram represents quantitation of western blots normalized to GAPDH (GDH). (B) Western blot for the cell cycle regulators indicated in CT or shP53 cells, 2 or 3 days postinfection; Cyc, cyclin. See also Figure S2A. (C) Bar histogram shows expression of p53 in CT or shP53 cells as measured by RT-PCR 2 days postinfection. See also Figure S2A. (D) Double immunostaining for p53 (red) and gH2AX (green) 3 days post- infection. Note that cells with low p53 tend to accumulate DNA damage (arrows) and vice versa. Nuclear DNA by DAPI (blue). See also Figure S5. Scale bar, 50 mm. (E) Distribution histogram shows number of foci of gH2AX per nucleus, as scored by immunofluorescence as in Figure 3D and image analyses. Left bar

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Disruption, Expressing, Infection, Quantitation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Double Immunostaining

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 4. Disruption of Endogenous p53 in Human Keratinocytes Causes Mitotic Slip- page and Loss of Clonogenic Potential (A) Percent of total or polyploid (polyp) HK infected with shP53 undergoing DNA synthesis (BrdU+) for the periods of time postinfection indicated, as quantitated by flow cytometry and normalized to HK infected with CTGFP (CT). See also Figure S2B. (B) Percent of cells in the G1/S (2N), G2/M (4N), or polyp phases of the cell cycle in CT (gray) or shP53 (light red) cells 3 days postinfection, as determined by flow cytometry. Red arrow shows the shift of shP53 cells toward polyploidy, a consequence of mitotic slippage. See also Fig- ures S2B and S2C. (C) Double immunostaining for K1 (green, arrow heads) and mitotic cyclin B (CycB, red; arrows). Nuclear DNA by DAPI (blue). shP53 cells dis- played frequent accumulation of cytoplasmic cy- clin B (cytopl+), and scoring is shown in the bar histogram. See also Movie S2. (D) Clonogenic capacity of CT or shP53 cells (10,000 cells plated 3 days postinfection). Bar and circle histograms show the number (bars) and percent (circles) of actively shP53 growing col- onies (light gray) versus small abortive, differenti- ated colonies (dark gray). Error bars are SEM of duplicate (A–C) or triplicate (D) samples of representative experiments. *p < 0.05.

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Disruption, Infection, DNA Synthesis, Cytometry, Double Immunostaining

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 5. Loss of Endogenous p53 in Human Keratinocytes Triggers Terminal Differenti- ation, Stratification, and Shedding (A) Dot plots show flow-cytometry analyses for GFP (green) versus light scatter (SSC-A; left) or involucrin (invol, right) of HK infected with shP53. Bar histograms show percent of differentiating (high light scatter; diff. in dot plot) or Invol positive (i+ in dot plot) cells in the GFP (+) or non-GFP () populations, as indicated. CT, cells infected with control vector. See also Figure S4A. (B) Double immunostaining for GFP (green) and Invol (red) 5 days postinfection of HK with CTGFP (CT) or shP53 as indicated; nuclear DNA by DAPI (blue). Bar histograms show percentage of cells positive for Invol and K1 (flow cytometry), or K10 (immunofluorescence) in shP53 cells relative to CT cells. Scale bar, 50 mm. See also Figure S4. (C) Expression of differentiation markers K1, ker- atin K10, filaggrin (Fil), and Invol, as determined by RT-PCR in shP53 cells relative to CT cells 2 days postinfections. (D) Snap frames from Movies S4 and S6 showing the accumulation of shP53 cells in mitosis (arrows) 5 days postinfection. Nuclear DNA by NucBlue (blue). Amplified insets show a cell blocked in metaphase (bottom panel: DNA only). See also Movies S4, S5, and S6. Bar histogram shows percent of shP53 cells shed into the medium, relative to control cells. Error bars are SEM of duplicate samples of repre- sentative experiments. *p < 0.05. Scale bars repre- sent 50 mm (B and D) or 10 mm (small insets in D).

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Cytometry, Infection, Control, Plasmid Preparation, Double Immunostaining, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 6. A Mutant Inactive Conformation of p53 Induces Keratinocyte Differentiation and Reduces the Great Proliferative Poten- tial Cell Compartment (A) Detection of p53 by western blotting in kerati- nocytes expressing control vector (CT) or the temperature-sensitive mutant p53 (p53ts) 48 hr after the temperature switch to 32C or 39C as indicated. Bar histogram show quantitation of p53 western blots normalized to GAPDH (GDH). Note that p53 accumulates at 39C (inactive conformation). (B) Top: immunofluorescence for p53 (green) on p53ts cells as in (A); Bottom: double immunofluo- rescence for involucrin (Invol, green) and p53 (red) on p53ts cells 4 days after the temperature switch. Note the binucleate differentiating p53ts cells that accumulate p53 at 39C (arrows). Nuclear DNA by DAPI (blue). See also Figure S7A. Scale bar, 50 mm. (C) Representative flow cytometry for the expres- sion of Invol (top) or the hyperproliferative marker keratin K16 (bottom) in p53ts cells 6 days after the temperature switch, as indicated. Bar histograms show percent of morphologically differentiated (high scatter) cells or cells positive for Invol or K16 as indicated, normalized to CT cells; quantitation of positive cells by the gates indicated on the histo- grams (+), as determined by a negative isotype antibody control (dotted line; CD8). See also Figure S7B. (D) Clonogenic capacity of p53ts cells plated 5 days after the 39C temperature switch. A total of 2,500 proliferative cells were plated. Bar and circle histogram show number (bars) or percent (circles) of actively shP53 growing colonies (light gray) versus small abortive, differentiated colonies (dark gray). Error bars are SEM of duplicate (C) or triplicate (D) samples of representative experiments. *p < 0.05.

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Mutagenesis, Western Blot, Expressing, Control, Plasmid Preparation, Quantitation Assay, Cytometry, Marker

Journal: Cell Reports Medicine

Article Title: Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities

doi: 10.1016/j.xcrm.2021.100240

Figure Lengend Snippet: p53 downregulation rescues neurodevelopmental defects in XLID JMS patient hiPSCs (A) Relative number of rosettes upon neural differentiation of WT-DYS0100, JMS-cl.1, and JMS-cl.1-expressing shRNA control (shCtrl) or shRNA targeting p53 (shP53a and shP53b) hiPSCs (n ≥ 3, biological replicates). JMS cells harbor HUWE1 p.G4310R. (B–D) mRNA expression levels of NES/NESTIN (B), TUBB3/TUJ1 (C), and DCX (D) analyzed by qRT-PCR in WT and JMS neural cells (collected at day 13) addressed by qRT-PCR. (E) Immunofluorescence analysis of the TUJ1 signal in WT-DYS0100, JMS-cl.1, JMS-cl.1 shCtrl, and JMS-cl.1 shP53a and shP53b at day 13 of neural differentiation. (F–H) qRT-PCR analysis of CDKN1A/p21 (F), GADD45α (G), and BAX (H) expression in WT and JMS neural cells (collected at day 13). All error bars indicate mean ± SEM (n = 3, biological replicates); one-way ANOVA followed by Dunnett’s post-test (A); one-tailed t test (B–D and F–H); ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, n.s. ≥ 0.05. See also and .

Article Snippet: JMS-cl.1 and JMS-cl.2 hiPSCs were transduced with lentiviruses encloding for the non-specific control short-hairpin RNA (shControl; pLKO.1 puro (Addgene ID: 8453)) or the p53 targeting shRNAs; shP53a (shp53 pLKO.1 puro shRNA (Addgene ID: 19119) ), shP53b (shp53 pLKO.1 puro shRNA-427 (Addgene ID: 25636) ) and shP53c (shp53 pLKO.1 puro shRNA-941(Addgene ID: 25637) ).

Techniques: Expressing, shRNA, Control, Quantitative RT-PCR, Immunofluorescence, One-tailed Test

Journal: Cell Reports Medicine

Article Title: Increased p53 signaling impairs neural differentiation in HUWE1-promoted intellectual disabilities

doi: 10.1016/j.xcrm.2021.100240

Figure Lengend Snippet:

Article Snippet: JMS-cl.1 and JMS-cl.2 hiPSCs were transduced with lentiviruses encloding for the non-specific control short-hairpin RNA (shControl; pLKO.1 puro (Addgene ID: 8453)) or the p53 targeting shRNAs; shP53a (shp53 pLKO.1 puro shRNA (Addgene ID: 19119) ), shP53b (shp53 pLKO.1 puro shRNA-427 (Addgene ID: 25636) ) and shP53c (shp53 pLKO.1 puro shRNA-941(Addgene ID: 25637) ).

Techniques: Virus, Recombinant, Knock-Out, Library Quantification, RNA Sequencing, Control, Mutagenesis, shRNA, Software, Reverse Transcription, SYBR Green Assay, Imaging