addgene plko 1 p53 shrna  (Addgene inc)

Journal: Stem Cell Reports

Article Title: ASCL1 promotes nuclear shrinkage in transdifferentiation by suppressing NUP37

doi: 10.1016/j.stemcr.2026.102823

Figure Lengend Snippet: Reduction of nuclear size during the direct conversion of human fibroblasts to neurons (A) Schematic for the transdifferentiation of human fibroblasts to iNs by lentiviruses expressing ASCL1, miR124-9-9 ∗ -BclxL, and p53 shRNA (AMp, uppercase for overexpression and lowercase for knockdown). –FBS, serum withdrawal to synchronize cell cycle at the G1/S checkpoint. Scale bar, 100 μm. (B) Phase contrast images of MRC5 cells under conversion at the indicated time points. Scale bar, 100 μm. Insets, super-resolution images of DAPI-stained nuclei. Scale bar, 10 μm. (C) Nuclear volume quantification for MRC5 cells throughout reprogramming. n = 50 frames from 3 independent experiments for each time point, unpaired t test vs. day −2. ∗ p < 0.01. (D) Quantification of nuclear area for MRC5 cells throughout reprogramming. n = 50 frames from 3 independent experiments for each time point, unpaired t test vs. day −2, ∗ p < 0.01. (E) Quantification of nuclear area at the indicated time points as MRC5, AG22056 newborn foreskin fibroblasts, or GM09918 (78 years) skin fibroblasts were being converted to iNs. n = 50 frames from 3 independent experiments for each time point, unpaired t test vs. day −2, ∗ p < 0.01. (F) The average area of MRC5, AG22056, or GM09918 cells as fibroblasts at day −2 (Fib) or TUJ1 + or MAP2 + iNs. ns, no significance. n = 50 frames from three independent experiments. (G) iPSC-derived cortical neurons were co-stained for MAP2 and DAPI at days 30, 40, and 80 of differentiation. Scale bar, 50 μm. Inset, super-resolution images of neuronal nuclei; scale bar, 10 μm. (H) Quantification of nuclear area of the indicated samples. ∗ p < 0.01, vs. the preceding bar (or D30 for iPSC-derived neurons), n = 50 frames from 3 independent experiments.

Article Snippet: We purchased the following plasmids from Addgene: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), pTight-9-124-Bclx (miR9/9 ∗ -124, #60857), pRL-SV40P (#27163), and pGL3 enhancer vector (#212938).

Techniques: Expressing, shRNA, Over Expression, Knockdown, Staining, Derivative Assay

Journal: Stem Cell Reports

Article Title: ASCL1 promotes nuclear shrinkage in transdifferentiation by suppressing NUP37

doi: 10.1016/j.stemcr.2026.102823

Figure Lengend Snippet: NUP37 knockdown significantly enhanced AMp-mediated transdifferentiation and nuclear shrinkage (A) Western blot of NUP37 in MRC5 cells transduced without (−) or with the indicated reprogramming factors. (B–D) MRC5 human fibroblasts reprogrammed with ASCL1, MIR124-9-9 ∗ -BclxL, and p53 shRNA (AMp) (B), AMp and NUP37 shRNA (AMpu) (C), or AMp and NUP37 overexpression (AMpU) (D) were co-stained as indicated on day 14. Scale bar, 100 μm. (E–G) Reprogramming efficiency (E) as measured by the percentages of TUJ1 + or MAP2 + cells among all DAPI + cells, reprogramming yield of MAP2 + cells per frame (F), and the number of DAPI + cells per frame (G) at day 14. # and ∗ , p < 0.05, n = 15 (3 experiments, 5 frames each), vs. AMp for the indicated cell type, unpaired t test. (H) Nuclear area of MAP2 + neurons for each condition. ∗ p < 0.001, n = 50 frames from 3 independent experiments, vs. AMp, unpaired t test. (I–P) MRC5 cells reprogrammed with AMp (I–L) or AMpu (M–P) were co-stained as indicated at different time points. Scale bar, 100 μm. (Q‒S) (Q) Reprogramming efficiency of MAP2 + -generated neurons per DAPI + nuclei. (R) Yield of MAP2 + neurons. (S) Number of DAPI + cells per frame. ∗ p < 0.01, n = 15 (3 experiments, 5 frames each), vs. AMp at the same time point, unpaired t test. (T) RT-qPCR measurement of mature neuronal markers in AMp- or AMpu-induced neurons at D14. ∗ p < 0.05, n = 6 (3 experiments, duplicate for each), vs. AMp, unpaired t test.

Article Snippet: We purchased the following plasmids from Addgene: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), pTight-9-124-Bclx (miR9/9 ∗ -124, #60857), pRL-SV40P (#27163), and pGL3 enhancer vector (#212938).

Techniques: Knockdown, Western Blot, shRNA, Over Expression, Staining, Generated, Quantitative RT-PCR

Journal: Stem Cell Reports

Article Title: ASCL1 promotes nuclear shrinkage in transdifferentiation by suppressing NUP37

doi: 10.1016/j.stemcr.2026.102823

Figure Lengend Snippet: Cooperation of ASCL1 and NUP37 shRNA in reprogramming and nuclear shrinkage (A–P) MRC5 human fibroblasts were reprogrammed without or with the indicated combinations of ASCL1 (A), miR124-9-9 ∗ -BclxL (M), p53 shRNA (p) and NUP37 shRNA (u), and co-stained as indicated at day 14. Bar, 100 μm. (Q–S) Reprogramming efficiency (Q) as measured by the percentages of TUJ1 + or MAP2 + cells among all DAPI + cells, reprogramming yield of MAP2 + cells per frame (R), and the number of DAPI + cells per frame (S) at day 14. # and ∗ , p < 0.001, n = 15 (3 experiments, 5 frames each), vs. the corresponding condition without u for the indicated cell type, unpaired t test. $ p < 0.001, n = 15 (3 experiments, 5 frames each), vs. no virus (−V), unpaired t test. (T) Nuclear area for each condition. ∗ p < 0.05, n = 50 frames from 3 independent experiments, vs. the corresponding condition without u; unpaired t test. $ p < 0.005, n = 50 frames from 3 independent experiments, vs. no virus (−V), unpaired t test.

Article Snippet: We purchased the following plasmids from Addgene: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), pTight-9-124-Bclx (miR9/9 ∗ -124, #60857), pRL-SV40P (#27163), and pGL3 enhancer vector (#212938).

Techniques: shRNA, Staining, Virus

Journal: Stem Cell Reports

Article Title: PTBP2 attenuation facilitates fibroblast to neuron conversion by promoting alternative splicing of neuronal genes

doi: 10.1016/j.stemcr.2023.09.012

Figure Lengend Snippet: PTBP2 knockdown enhances the direct conversion of human skin fibroblasts to neurons (A−Q) Using the reprogramming strategy in (A), AG22056 primary foreskin fibroblasts were reprogrammed to neurons in neural induction medium without (B) or with the indicated combinations of lentiviruses (C−Q) expressing ASCL1 (A), MiR9/9 ∗ -124 (M), PTBP2 shRNA (n), and p53 shRNA (p). Cells at day 14 were stained for MAP2, TUJ1, and DAPI (B−Q). Scale bar, 100 μm. (R−T) Reprogramming yield as defined by the number of MAP2 + , TUJ1 + , and DAPI + cells per frame of view (R, T), and reprogramming efficiency as defined by the ratio of MAP2 + /DAPI + cells or TUJ1 + /DAPI + cells (S) were quantified. n = 15 frames from 3 independent experiments for each condition. Data are represented as mean ± SD, ∗ p < 0.05, ∗∗∗ p < 0.001. ns, non-significant, one-way ANOVA with Sidak test. The difference between AMp and AMnp is highlighted with a red line and red asterisks to indicate statistical significance. For Figure 1 T, statistical significance was compared with the control group. (U−V) Electrophysiological recordings showed that the neurons at day 40 had voltage-gated Na + and K + currents (U), as well as evoked action potentials in response to current injections (V).

Article Snippet: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), and pTight-9-124-BclxL (miR9/9 ∗ -124, #60857) were from Addgene.

Techniques: Knockdown, Expressing, shRNA, Staining, Control

Journal: Stem Cell Reports

Article Title: PTBP2 attenuation facilitates fibroblast to neuron conversion by promoting alternative splicing of neuronal genes

doi: 10.1016/j.stemcr.2023.09.012

Figure Lengend Snippet: A sequential model for the transdifferentiation of human skin fibroblasts to neurons (1) Synchronization of the cell cycle, by serum withdrawal, at the G1/S checkpoint makes the cells receptive to transdifferentiation. (2) The pioneer transcription factor ASCL1 causes cell-cycle exit and induces neuronal genes. (3) Knockdown of p53 induces the TET family of DNA demethylases to facilitate the epigenetic reprogramming. (4) MIR-124 promotes the conversion through pleiotropic actions that include the suppression of REST and the reduced expression of nPTB via nonsense-mediated decay. (5) nPTB knockdown and the induction of RBFOX3 during the conversion work in concert to drive a neuronal AS program that improves the transdifferentiation.

Article Snippet: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), and pTight-9-124-BclxL (miR9/9 ∗ -124, #60857) were from Addgene.

Techniques: Knockdown, Expressing

Journal: bioRxiv

Article Title: PITAR , a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.1101/2023.04.11.536370

Figure Lengend Snippet: A. Genomic track for PITAR derived from ChIRP-RNA sequencing using Odd, Even, and LacZ antisense probe. B. PITAR Pulldown by ChIRP assay was quantified using qRT-PCR. C. The Venn diagram represents the association of four datasets (ChIRP enriched genes, PITAR positive correlated genes from TCGA, GBM vs. normal upregulated genes from TCGA, and GSC vs. DGC upregulated genes from GSE54791). D. The selected fifteen genes from the Venn diagram are plotted in the scatter plot, and an arrow marked TRIM28 as a selected target. E. The volcano plot depicts up-regulated (n=420) and down-regulated (n=526) genes upon PITAR knockdown compared to siNT. The gene expression matrix between siPITAR and siNT was used to construct a volcano plot to visualize differentially expressed genes. F. Gene set enrichment analysis (GSEA) of differentially regulated genes was performed based on PITAR expression level at log2fold >0.58 and p<0.05. G. The GSEA plots depict the enrichment of p53 up and down target genes results derived from PITAR silencing in U87 cells. Data are shown as mean ± SD (n=3). *** P -value <0.001, ** P -value <0.01, * P -value <0.05.

Article Snippet: The shRNA plasmid (pLKO.1) for p53 was obtained from the TRC library (Sigma, IISc).

Techniques: Derivative Assay, RNA Sequencing Assay, Quantitative RT-PCR, Knockdown, Expressing, Construct

Journal: bioRxiv

Article Title: PITAR , a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.1101/2023.04.11.536370

Figure Lengend Snippet: A. Relative expression of PITAR, TRIM28, TP53, and CDKN1A was quantified by qRT-PCR in PITAR-silenced U87 cells compared to siNT. B. The protein expression of TRIM28, p53, and p21 was measured by immunoblotting in PITAR-silenced U87 cells compared to siNT. C & D. Cells were transfected with pcDNA-PITAR (PITAR OE)/vector control plasmid (pcDNA) and harvested 48h post-transfection for qRT-PCR (PITAR, TRIM28, TP53, and CDKN1A) or immunoblotting with indicated antibodies (TRIM28, p53, and p21). GAPDH served as the control. E. The Immunoblotting Half-life of p53 protein in PITAR-silencing (siPITAR) and control (siNT) U87 cells was measured, and Immunoblotting assays were used to detect p53 in U87 cells with the treatment of cycloheximide (CHX; 50 μg/mL). The relative expression of the remaining TRIM28 was plotted using linear regression after normalizing to the 0th hour of the respective condition. F. The endogenous level of p53 ubiquitination was measured in pcDNA-PITAR (PITAR OE)/vector plasmid (pcDNA) U87 cells by p53 immunoprecipitation followed by immunoblotting with the indicated antibodies in the presence and absence of MG132. G. The PG13-Luc activity was measured in PITAR-silenced/siNT U87 cells, which were further transfected with TRIM28 overexpression/Vector control plasmid. H. The relative expression of CDKN1A was measured by qPCR in PITAR-silenced/siNT U87 cells, which were further transfected with TRIM28 overexpression/Vector control plasmid. I. The protein expression of TRIM28, p53, and CDKN1A was measured by immunoblotting in PITAR-silenced/siNT U87 cells, which were further transfected with TRIM28 overexpression/Vector control plasmid. J. The relative expression of CDKN1A was measured in the presence and absence of adriamycin by qPCR in PITAR OE/VC U87 cells. K. The protein expression of TRIM28, p53, ac-p53, and CDKN1A was measured in the presence and absence of adriamycin by immunoblotting in PITAR OE/VC U87 cells. GAPDH served as the control. L. The Cell cycle analysis was performed in the presence and absence of Adriamycin in PITAR OE/VC U87 cells. M. The relative expression of PITAR, TRIM28, and CDKN1A was measured in the presence and absence of adriamycin by qPCR in U87 cells. N. The relative expression of PITAR and TRIM28 was measured in the presence and absence of Adriamycin/CGK733 using qPCR in U87 cells. O. TRIM28 protein expression was measured by immunoblotting in the presence of Adriamycin/CGK733 in U87 cells. P. The relative expression of PITAR and TRIM28 was measured by qRT-PCR in PITAR-silenced U87 cells after Adriamycin treatment. Data are shown as mean ± SD (n=3). *** P -value <0.001, ** P -value <0.01, * P -value <0.05.

Article Snippet: The shRNA plasmid (pLKO.1) for p53 was obtained from the TRC library (Sigma, IISc).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Activity Assay, Over Expression, Cell Cycle Assay

Journal: bioRxiv

Article Title: PITAR , a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.1101/2023.04.11.536370

Figure Lengend Snippet: A. The bright field image represents RG5 sphere growth on siNT/siPITAR condition. B&C. RG5 sphere growth was quantified by the measurement of sphere count and sphere size using ImageJ ( https://imagej.nih.gov/ij/download.html ). D. Limiting dilution assay was performed in RG5 cells. The graph represents the percentage of wells without spheres as a function of the number of PITAR Knockdown cells compared to control cells. E. Relative expression of PITAR, TRIM28, TP53, and CDKN1A was quantified by qRT-PCR in PITAR-silenced RG5 cells compared to siNT. F. The bright field image represents RG5 sphere growth on PITAR OE compared to the control vector. G&H. RG5 sphere growth was quantified by measuring sphere count and size in the PITAR OE condition compared to the control vector using ImageJ (). I. Relative expression of PITAR, TRIM28, TP53, and CDKN1A was quantified by qRT-PCR in PITAR OE condition of RG5 cells compared to the control vector. Data are shown as mean ± SD (n=3). *** P -value <0.001, ** P -value <0.01, * P -value <0.05.

Article Snippet: The shRNA plasmid (pLKO.1) for p53 was obtained from the TRC library (Sigma, IISc).

Techniques: Limiting Dilution Assay, Knockdown, Control, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: Knockdown of DNAJA1 specifically reduces protein levels of conformational mutp53, but not DNA contact mutp53 and wtp53. ( A , B ) Western blotting for p53, DNAJA1, and GAPDH ( A ) and immunofluorescence for p53, DNAJA1, and DAPI ( B ) using multiple cancer cells with different p53 status with or without knockdown of DNAJA1 (JA1) and p53 (p53). Scale bar: 50 µm.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Knockdown, Western Blot, Immunofluorescence

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: Identification of a compound that binds DNAJA1 to specifically reduce conformational mutp53. ( A ) Chemical structure of PLIHZ compound, derived from plumbagin, which was identified through a molecular docking ( left ). Ribbon and crystal structures of DNAJA1 protein (PDB: 2LOI) showing binding of DNAJA1 to PLIHZ at tyrosine residue Y7 ( right ) with a 2.6 Å bond distance and binding free energy of −6.6 kcal/mol. ( B ) CETSA showing intracellular binding of PLIHZ to DNAJA1. A representative Western blotting for DNAJA1 and GAPDH using protein extracts from CAL33 (p53 R175H ) cells with treatment with DMSO and PLIHZ at 80 µM for 4 h, followed by incubation at different temperatures for 3 min ( left ). A representative blot using protein extracts from unheated cells are also shown. A summarized graph showing normalized DNAJA1 band densities at different temperatures of 40, 43, 46, 49, 52, 55, and 58 °C ( right ). Mean ± SEM from three independent experiments ( n = 3). ** p < 0.01 for two-way ANOVA. ( C ) Western blotting for p53, DNAJA1, and GAPDH using indicated cells with different p53 status, treated with DMSO or PLIHZ at ~1/2 of 24h-IC50 for 24 h. ( D ) Immunofluorescence for p53, DNAJA1, and DAPI using indicated cells treated with DMSO or PLIHZ at ~1/2 of 24h-IC50 for 24 h.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Derivative Assay, Binding Assay, Residue, Western Blot, Incubation, Immunofluorescence

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: PLTFBH, an analog of PLIHZ, specifically reduces conformational mutp53 levels similar to PLIHZ. ( A ) Chemical structures of PLIHZ analogs including PLIHZ, PLFBH, PLTFBH, PLFUH, and PLOCT. ( B ) Western blotting for p53 and GAPDH using HN31 cells treated with different PLIHZ analogs (40 µM for 24 h). ( C ) Western blotting for p53, DNAJA1, and GAPDH using HN31, MDA-MB-231, and U2OS cells treated with PLTFBH at ~1/2 of 24h-IC 50 . ( D ) Immunofluorescence for p53, DNAJA1, and DAPI using HN31, MDA-MB-231, U2OS, and H1299 cells treated with PLTFBH at ~1/2 of 24h-IC 50 . Scale bar: 50 µm. ( E ) CETSA showing intracellular binding of PLTFBH to DNAJA1 in CAL33 cells: a representative Western blotting for DNAJA1 and GAPDH using protein extracts from HN31 cells ( left ); a summarized graph showing normalized DNAJA1 band densities at different temperatures ( right ). Mean ± SEM from three independent experiments ( n = 3). * p < 0.05 for two-way ANOVA. ( F ) Western blotting for p53, DNAJA1, and GAPDH using HN31 cells with or without knockout for DNAJA1 ( JA1 ) or p53 ( p53 ) using the CRISPR-Cas9 strategy. ( G ) Summary of MTT assays using control, DNAJA1 knockout, or p53 knockout HN31 treated with different concentrations of PLTFBH for 72 h. Mean ± SEM from three independent experiments ( n = 3). n.s.: not significant for one-way ANOVA. IC 50 values of PLTFBH for each sub-cell line are shown on the right.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Western Blot, Immunofluorescence, Binding Assay, Knock-Out, CRISPR, Control

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: PLTFBH inhibits migratory potential of cancer cells in a manner dependent on DNAJA1 and conformational mutp53. ( A ) Transwell migration assays using indicated cells with different p53 status treated with PLTFBH at ~1/2 IC 50 for 12 h. All cells were pre-treated with PLTFBH for 12 h, followed by trypan blue staining and transwell migration assays. Top : summarized graphs. Bottom : representative images. Mean ± SEM from three independent experiments ( n = 3). *** p < 0.001 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 100 µm. ( B ) F-actin staining showing inhibition of filopodia formation in HN31 cells, but not MDA-MB-231, U2OS, and H1299 cells, by PLTFBH (TF). Top: summarized graph. Bottom : representative images. Mean ± SEM from three independent experiments ( n = 3). **** p < 0.0001 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 10 µm. ( C ) Transwell migration assays using DNAJA1- or p53 -knockdown HN31 cells treated with PLTFBH at ~1/2 IC 50 for 12 h. Cells were pre-treated with PLTFBH for 12 h. Mean ± SEM from three independent experiments ( n = 3). ** p < 0.01 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 100 µm. ( D ) Rac1/Cdc42 activation assays following pulldown of active Rac1 and Cdc42 using protein extracts from HN31 cells treated with DMSO (D) or PLTFBH (TF) at ~1/2 of 24h-IC 50 . Left : representative immunoblots. Right : summarized graph. Mean ± SEM from three independent experiments ( n = 3). * p < 0.05, *** p < 0.001 for two-tailed Student’s t -test.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Migration, Staining, Two Tailed Test, Inhibition, Knockdown, Activation Assay, Western Blot

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: Mutations at Y7 and Y8 residues in DNAJA1 abrogate the ability of PLTFBH to deplete DNAJA1 and conformational mutp53. ( A ) Western blotting to detect exogenous DNAJA1 and GAPDH, as well as endogenous p53 (p53 C176F ), using DNAJA1 -knockout HN31 cells (JA1KO) expressing exogenous wild-type (wt), Y7A mutant (Y7A), and Y8A mutant (Y8A) DNAJA1, treated with DMSO (D) or PLTFBH (TF) at ~1/2 IC 50 for 24 h. ( B ) Immunofluorescence for p53, DNAJA1, and DAPI using the same experimental set of HN31 sub-cell lines as in A. Scale bar: 50 µm. ( C ) F-actin staining using the HN31 sub-cell lines treated with DMSO or PLTFBH at ~1/2 IC 50 for 24 h. Mean ± SEM from three independent experiments ( n = 3). ** p < 0.01 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 10 µm. Left : representative images. Right : summarized graph.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Western Blot, Knock-Out, Expressing, Mutagenesis, Immunofluorescence, Staining, Two 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: p53 signaling is hyperactivated in cells from XLID patients with mutated HUWE1 (A) Schematic representation of XLID-causative HUWE1 mutations analyzed in this study (p.R2981H, p.R4187C, and JMS-p.G4310R; HUWE1 duplication: 2×). Depicted HUWE1 domains: ARLD1/2, Armadillo repeat-like domains 1/2; UBA, ubiquitin-association domain; WWE, tryptophan-tryptophan-glutamate domain; BH3, Bcl-2 homology 3 domain; HECT, homologous to E6-AP carboxyl terminus domain. (B) Top five most significant KEGG pathway terms as determined by gene set enrichment analysis (GSEA) of common differentially expressed genes in XLID patient-derived lymphoblastoid cells (LCs) compared with healthy individual cells (Benjamini corrected p < 0.05). (C) Heatmap of common differentially expressed genes in XLID compared with healthy LCs belonging to the KEGG p53 signaling term. (D–F) mRNA levels of p53 target genes CDKN1A/p21 (D), GADD45α (E), and BBC3/PUMA (F) determined by qRT-PCR analysis of four healthy (GM03798, GM07535, GM16113, and GM16119) and five XLID LCs with mutated HUWE1 (p.R2981H, p.R4187C, JMS-p.G4310R, and HUWE1 duplication). All error bars indicate mean ± SEM (n = 3, biological replicates). Two-tailed unpaired t test; ∗∗p ≤ 0.01, ∗∗∗∗p ≤ 0.0001.

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: Ubiquitin Proteomics, Derivative Assay, Quantitative RT-PCR, Two 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: p53 accumulation and activation, caused by HUWE1 p.G4310R, perturb the cell cycle and impair proliferation in JMS patient-derived cells (A) Immunoblot analysis of the HUWE1, p53, and p21 protein levels in healthy control LCs and LCs from two JMS patients (JMS1 and JMS2). p53 protein levels relative to tubulin, serving as loading control, are indicated. (B) In vitro ubiquitination of purified recombinant p53 with increasing amounts of wild-type (WT) and p.G4310R HECT proteins. (C) In silico analysis of difference in the folding free energy change (ΔΔG) of WT and G4310R HUWE1 using the indicated prediction tools. (D) Cell-cycle distribution determined by flow cytometry of healthy control, JMS1, and JMS2 LCs. (E) Fraction of annexin V-positive apoptotic cells measured by flow cytometry. (F) Proliferation rate of healthy control, JMS1, and JMS2 LCs. All error bars indicate mean ± SEM (n ≥ 3, biological replicates). Statistical significance was determined by two-way ANOVA with Tukey post-test (D) and Bonferroni post-test (F); one-way ANOVA with Bonferroni post-test; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, n.s. ≥ 0.05. See also Figure S1 .

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: Activation Assay, Derivative Assay, Western Blot, Control, In Vitro, Ubiquitin Proteomics, Purification, Recombinant, In Silico, Flow Cytometry

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: Neural differentiation of JMS patient hiPSCs is impaired and accompanied by activation of p53 signaling (A) Representative bright-field images of neural differentiation of healthy control hiPSCs (WT-DYS0100 and WT-CRL(S)23) and two hiPSC clones from a JMS patient expressing HUWE1 p.G4310R (JMS-cl.1 and JMS-cl.2) at day 11; neural rosette structures are visible. (B) Relative number of rosettes formed in WT and JMS clones (n ≥ 3, biological replicates). (C–F) qRT-PCR analysis of gene expression of OCT4 (C), NES/NESTIN (D), TUBB3/TUJ1 (E), and DCX (F) in WT and JMS hiPSCs and neural cells (collected at day 13) (n = 3, biological replicates). (G) Immunofluorescence analysis of TUJ1 in WT and JMS hiPSCs at day 13 of neural differentiation. (H) Relative intensity of the TUJ1 signal in WT and JMS clones from experiments like the one in (G) (n ≥ 2, biological replicates). (I–L) qRT-PCR analysis of mRNA levels of the p53 target genes CDKN1A /p21 (I), GADD45α (J), BBC3/PUMA (K), and BAX (L) in WT and JMS hiPSCs and neural cells (collected at day 13) (n = 3, biological replicates). All error bars indicate mean ± SEM; one-way ANOVA followed by Tukey post-test (B); two-way ANOVA followed by Tukey post-test (C–F and I–L); ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, n.s. ≥ 0.05. Scale bar: 400 μm. 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: Activation Assay, Control, Clone Assay, Expressing, Quantitative RT-PCR, Gene Expression, Immunofluorescence

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