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