p21 coding sequence  (Addgene inc)

Journal: iScience

Article Title: GPX4-dependent ferroptosis sensitivity is a fitness trade-off for cell enlargement

doi: 10.1016/j.isci.2025.112363

Figure Lengend Snippet: Increased lipid peroxidation in large cells (A) Digital holography images of RSL3 treatment time course for control, doxorubicin-treated, and palbociclib-treated RPE1 cells. (B) Quantification of imaging parameters at 0 and 4 h after RSL3 addition. The blue numbers in the optical volume indicate the increase in cell volume (swelling) between 0- and 4-h time points. (C) Lipid peroxidation in RPE1 cells using C11-bodipy lipid peroxidation sensor and flow cytometry. Probe oxidation results in a shift of the fluorescence emission peak from red (PE-A) to green (FITC-A) channel. Cells treated with palbociclib were treated with or without 1 μM RSL3 and 1 μM Fer-1 as indicated. Data shown are mean ± SD, n = 3. (D and E) (D) Same as (C) but cells were treated with 50 nM doxorubicin. Statistical analysis in (C and D) was ANOVA followed by Tukey’s test. (E) Digital holographic quantification of cell areas and phase shifts for single GPX4 KO cells treated with (blue line) or without (red line) doxorubicin for 3 days after which they were imaged for 6 h in the presence of ferroptosis inhibitor Fer-1. (F) Same treatment as in (E), but Fer-1 was washed out immediately before imaging. Mean values are in solid line, with error bars showing standard deviation; between 52 and 660 cells were tracked. (G) Tetracycline-inducible expression of mGreenLantern-p21. The cell size distribution with and without Tet-induction for 3 days was measured with coulter counter. (H) WB analysis of mGreenLantern-p21 with and without Tet-induction for 3 days, followed by 100 nM RSL3 for 24 h. (I) Senescence-associated β-galactosidase staining of mGreenLantern-p21 cells. Data shown are mean ± SD, n = 3. ANOVA with Tukey’s test. See also Figure S3 .

Article Snippet: The RPE1 mGreenLantern-p21 cells were established by cloning mGreenLantern in frame with p21 coding sequence into AAVS1-Tet plasmid (Addgene plasmid #158663, a kind gift from Masato Kanemaki).

Techniques: Control, Imaging, Flow Cytometry, Fluorescence, Standard Deviation, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: Effects of circPCNX on p21 mRNA and p21 protein. (A–D) AUF1 binding to p21 mRNA (A), p21 mRNA stability (B), p21 mRNA levels (C), and p21 protein levels (D) were assessed after transfection with pcDNA3(EV) or with pcDNA-circPCNX to overexpress circPCNX. (A) AUF1 binding to circPCNX, PCNX mRNA and p21 mRNA was assessed one day after transfection; values were first normalized to the levels of a transcript (GAPDH mRNA) that encodes a housekeeping protein, and afterwards normalized to the RNA levels in the IgG IP. (B) One day after transfection, cells were treated with Actinomycin D to block de novo transcription for the times indicated, and the time required for p21 mRNA to reach 50% of its initial abundance (the half-life) in each transfection group was assessed by RT-qPCR analysis. A stable transcript (ACTB mRNA) was included as control. At one and three days after transfection of plasmids, changes in the levels of p21 mRNA (C) or p21 protein (D) were assessed by RT-qPCR and western blot analyses, respectively. (E–H) AUF1 binding to p21 mRNA (E), p21 mRNA stability (F), p21 mRNA levels (G), and p21 protein levels (H) were assessed after transfection with Ctrl siRNA or circPCNX siRNA to silence circPCNX. (E) AUF1 binding to circPCNX, to PCNX mRNA and to p21 mRNA was assessed one day after transfection; as above, values were first normalized to GAPDH mRNA, and afterwards normalized to RNAs in the IgG IP. (F) One day after transfection, cells were treated with Actinomycin D to block de novo transcription for the times indicated, and the time required for p21 mRNA to reach 50% of its initial abundance (the half-life) in each transfection group was assessed by RT-qPCR analysis. A stable transcript (ACTB mRNA) was included as control. At one and three days after siRNA transfections, changes in the levels of p21 mRNA (G) or p21 protein (H) were assessed by RT-qPCR and western blot analyses, respectively. Data in (A–C, E–G) represent the mean values ± SD from three biological replicates. Significance was established using Student's t-test. ** P ≤ 0.01 or *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Binding Assay, Transfection, Blocking Assay, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: AUF1 binding to p21 mRNA is attenuated by circPCNX, but not by mutant circPCNX unable to bind AUF1. (A) Twenty-four hours after transfecting HeLa cells with the plasmids shown, the association of p21 mRNA (or ACTB mRNA, in control reactions) with AUF1 was measured by RIP followed by RT-qPCR analysis and represented as ‘fold enrichment’. (B, C) In HeLa cells transfected with the plasmids indicated, the levels of p21 mRNA (B) were assessed 3 days after transfection by RT-qPCR analysis, and the levels of p21 protein (C) were assessed one and three days after transfection by western blot analysis, including GAPDH as loading control. (D) At the times shown following transfection of HeLa cells with the plasmids indicated, cell proliferation in each transfection group was assessed by using BrdU incorporation analysis. Data in (A, B, D) represent the mean values ± SD from four biological replicates. Significance was established using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Binding Assay, Mutagenesis, Quantitative RT-PCR, Transfection, Western Blot, BrdU Incorporation Assay

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: Expression levels of p21 mRNA and p21 protein are attenuated by circPCNX, but not by a mutant circPCNX unable to bind AUF1. (A) Reporter plasmids were created using the backbone of the psiCHECK2 dual luciferase reporter by inserting the p21 3′UTR (gray) with either the full-length wild-type (3′wt) sequence or with a mutation (3′del) in the AUF1-binding site (red). Vectors using the pCMV6 backbone were also created to express p21 protein from mRNAs that had the intact 3′UTR (3′wt) or had a mutation in the AUF1-binding site (3′del). (B) Forty-eight hours after transfecting HeLa cells with either pcDNA3(EV) or pcDNA3-AUF1 or with the siRNAs shown, the reporter plasmids psiCHECK2-p21-3′wt or psiCHECK2-p21-3′del were transfected and 24 h after that, luciferase activities (RL/FL) in each transfection group were assessed. (C) Twenty-four hours after transfecting HeLa cells with the plasmids indicated, the levels of p21 mRNA (with 3′wt or 3′del 3′UTRs) were measured by RT-qPCR analysis and normalized to neo mRNA expressed from the same plasmids; the levels of control ACTB mRNA were quantified in the same reactions; the levels of p21 protein in each transfection group were assessed by western blot analysis. (D) Ctrl or circPCNX siRNAs were transfected along with the plasmids shown (left, p21 3′wt; right, p21 3′del), and the rates of proliferation were assessed by measuring BrdU incorporation at the times indicated. (E) Summary model. Top, when circPCNX accumulates in cells, it can sequester AUF1 away from its target mRNAs, notably p21 mRNA, in turn allowing p21 mRNA stabilization, increased p21 expression, and growth suppression. Bottom, when circPCNX levels decline, or if circPCNX is unable to bind AUF1 (mutated or truncated), then AUF1 associates with p21 mRNA, reducing p21 mRNA stability and p21 levels, and enhancing cell proliferation. Image was created using BioRender. Data in (B–D) represent the mean values ± SD from four biological replicates. Significance was established using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Expressing, Mutagenesis, Luciferase, Sequencing, Binding Assay, Transfection, Quantitative RT-PCR, Western Blot, BrdU Incorporation Assay