p21 waf1 cip1  (Cell Signaling Technology Inc)

Journal: PLOS One

Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma

doi: 10.1371/journal.pone.0342767

Figure Lengend Snippet: (A) The effect of Myc-siRNA on AP4 mRNA expression was evaluated. (B) A time-course analysis showed a correlation between Myc and AP4 protein expression. (C) Downregulation of AP4 led to upregulation of p21. (D) AP4 regulates p21 mRNA expression. The results are represented as the mean ± SEM (n.s., no significance). **, p < 0.01 versus the related control groups. (E) A time-course analysis of AP4 KD showed that p21 expression increased inversely with AP4 levels, while Myc remained unaffected. (F) Changes in p21 expression influenced the phosphorylation of CDK2 and Rb. It was found that p21 reduces phosphorylation of both CDK2 and Rb. (G) The presence of p21 inhibits binding between CDK2 and CCNE.

Article Snippet: AP4-siRNA (Cat. #sc-37690) and p21-siRNA (Cat. #sc-29427), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Control, Phospho-proteomics, Binding Assay

Journal: Cell Death & Disease

Article Title: Cellular adaptations impact the biological activity of naphthalene diimide G-quadruplex ligands in ALT-positive osteosarcoma cells

doi: 10.1038/s41419-025-07908-2

Figure Lengend Snippet: A Representative photomicrographs showing the detection of ROS upon H 2 DCFDA staining of living U-2 OS and Saos-2 cells both untreated (UNT) and after a 1-day exposure to NMe2 or QN-302. Hydrogen peroxide (H 2 O 2 ) in the absence or presence of N-acetylcysteine (NAC) was used as positive and negative controls for ROS detection, respectively. Nuclei were counterstained with Hoechst 3342. Scale bars: 100 μm; magnification: × 10; B Representative photomicrographs showing mitochondrial morphological alterations in NMe2-treated vs. untreated U-2 OS and Saos-2 cells probed with an anti-COX IV antibody (green). Nuclei were counterstained with DAPI. Merged images are shown; scale bar: 10 μm; magnification: × 60; C Representative western immunoblotting showing p21 Waf1 protein amounts in untreated U-2 OS cells and after a 2-day exposure to either G4L (IC 50 ). β-tubulin was used to ensure equal protein loading. Cropped images of selected proteins are shown. The graph on the bottom reports the quantification of p21 Waf1 protein amounts in the indicated samples. Data have been reported as relative protein amounts with respect to β-tubulin and represents mean values ± s.d. ( N = 3). *** p < 0.001; **** p < 0.0001 (two tailed unpaired t -test); D Quantifica t ion of the total antioxidant capacity (TAC) in untreated (UNT) U-2 OS and Saos-2 cells and after a 2-day exposure to equitoxic (IC 50 ) amounts of NMe2 or QN-302. Data have been reported as the total antioxidant capacity, expressed as μM copper-reducing equivalents (μM CRE), according to the manufacturer’s instructions. Bars represent mean values ± s.d. ( N = 4); * p < 0.05 (two-tailed Mann-Whitney test); E Quantification of the Nrf2 binding activity to ARE sequences in untreated U-2 OS cells and after a 2-day exposure to either G4L (IC 50 ). Data have been reported as OD read at 450 nm in tested samples in the presence of wild-type (wt) or mutated (mut) ARE-containing consensus sequence used to test for binding competition. C + : internal positive control for Nrf2 binding activity provided with the kit. Bars represent mean values ± s.d. ( N = 4); * p < 0.05 (two-tailed Mann–Whitney test); F Assessment of NQO1 mRNA levels in untreated U-2 OS and Saos-2 cells and after a 2-day exposure to either G4L (IC 50 ). RT-qPCR data were reported as 2 −ΔCt and represents mean values ± s.d. ( N = 3). ** p < 0.01; *** p < 0.001; **** p < 0.0001 (two tailed unpaired t -test). GAPDH was used as normalizer; G Representative western immunoblotting showing the amounts of the Nrf2 downstream target NQO1 in untreated U-2 OS and Saos-2 cells and after a 2-day exposure to either G4L (IC 50 ). β-tubulin was used to ensure equal protein loading. Cropped images of selected proteins are shown. The graph at the bottom reports the quantification of NQO1 protein amounts in the indicated samples. Data have been reported as relative protein amounts with respect to β-tubulin and represents mean values ± s.d. ( N = 3). * p < 0.05; ** p < 0.01 (two tailed unpaired t -test); H Assessment of cell growth kinetics in siCTR (•)- and siNrf2 (▲)-transfected U-2 OS cells either untreated (blue) or after a 2-h exposure (pulse) to subtoxic amounts of NMe2 (green) or QN-302 (red). Data have been reported as the percentage of phase image confluency (determined by Incucyte® SX5 Live-Cell Imaging and Analysis System) normalized to the first time point (T 0 ) using the normalization function in GraphPad and represent mean values ± s.d. ( N = 4); *** p < 0.001 (2-way ANOVA).

Article Snippet: Target-specific siRNAs designed to silence the expression of TP53 (sip53; sc-29435); CDKN1A (sip21; sc-44214), BLM (siBLM; sc-29808) and NFE2L2 (siNrf2; sc-44332) as well as a control siRNA (siCTR; sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX). siRNAs were dissolved in RNase-free water to make a 10 μM stock solution, stored at −20 °C and diluted to obtain a final concentration of 50 nM immediately before use.

Techniques: Staining, Western Blot, Two Tailed Test, MANN-WHITNEY, Binding Assay, Activity Assay, Sequencing, Positive Control, Quantitative RT-PCR, Transfection, Live Cell Imaging

Journal: International journal of molecular sciences

Article Title: p21-Dependent Senescence Induction by BMP4 Renders Glioblastoma Cells Vulnerable to Senolytics.

doi: 10.3390/ijms26093974

Figure Lengend Snippet: Figure 3. Cell-size enlargement and senescence-like induction by BMP4 is dependent on p21. (A–H) Wild-type and p21 knockout (p21-KO) MES-like clone 3065-c475 cells, untreated or treated for 12–14 days with BMP4. (A) Western blot using antibodies against p21, lamin B1, cyclin D1, SOX2,

Article Snippet: For β-gal co-staining with p21 Waf1/Cip1, CellEvent Senescence-stained cells were blocked and permeabilized (5% FBS/0.5% saponin or in 5% BSA/0.25% Triton) for 15–30 min, followed by p21 Waf1/Cip1 antibody (dilution 1:500, #2947, Cell Signaling) staining in 1%BSA/PBS, 30 min in room temperature.

Techniques: Knock-Out, Western Blot

Journal: International journal of molecular sciences

Article Title: p21-Dependent Senescence Induction by BMP4 Renders Glioblastoma Cells Vulnerable to Senolytics.

doi: 10.3390/ijms26093974

Figure Lengend Snippet: Figure 4. Senolytic treatment eradicates the senescent-like cells via apoptosis. (A) Cancer Dependency Map (DepMap) analysis of AUC levels of drugs and compounds and CDKN1A gene expression. Senolytic drugs are indicated in yellow. (B) The correlation of CDKN1A expression and navitoclax sensitivity in cancer cell lines. Gliomas are indicated in yellow. (C,D) MES-like 3065-c475 cells treated

Article Snippet: For β-gal co-staining with p21 Waf1/Cip1, CellEvent Senescence-stained cells were blocked and permeabilized (5% FBS/0.5% saponin or in 5% BSA/0.25% Triton) for 15–30 min, followed by p21 Waf1/Cip1 antibody (dilution 1:500, #2947, Cell Signaling) staining in 1%BSA/PBS, 30 min in room temperature.

Techniques: Gene Expression, Expressing

Journal: Cell death & disease

Article Title: Therapy-induced senescence of glioblastoma cells is determined by the p21 CIP1 -CDK1/2 axis and does not require activation of DREAM.

doi: 10.1038/s41419-025-07651-8

Figure Lengend Snippet: Fig. 1 TMZ-induced cell death and senescence. A–D LN229 cells were exposed to different concentrations of TMZ for 144 h (A, B) or to 50 µM TMZ for different time periods (C, D). Experiments were performed in triplicates. A, C Cell death was measured by flow cytometry using PI staining. B, D Frequency of senescent cells was detected microscopically by SA-β-Gal staining. E, F LN229 cells were transfected with p21CIP1 specific siRNA or nonspecific siRNA and 24 h later exposed to 50 µM TMZ for 144 h. Cell death was measured by flow cytometry using PI staining (E) and frequency of senescent cells was detected by flow cytometry using C12FDG staining (F). G–I LN229 cells were exposed to 50 µM TMZ for different time periods. G Proliferation was measured by cell counting. H DNA synthesis was measured by BrdU assay. I DNA content per cell was measured using the NanoDrop 1000 Spectrophotometer. J LN229 cells were exposed to 50 µM TMZ for 144 h, and cell cycle distribution was measured by flow cytometry using PI staining. A representative experiment is shown. B, D, I Differences between treatment and control were statistically analyzed using Student’s t test (p < 0.001). F Differences between treatment and control, as well as between ns-si/TMZ and p21-si/TMZ were statistically analyzed using Student’s t test (**p < 0.01***p < 0.001).

Article Snippet: For gene silencing, predesigned siRNAs specific for p21CIP1 (sc-29427, Santa Cruz Biotechnology) were used; control human non-silencing siRNA (Silencer Select Predesigned siRNA Negative Control #1 siRNA; Ambion) was used as negative control.

Techniques: Cytometry, Staining, Transfection, Cell Counting, DNA Synthesis, BrdU Staining, Spectrophotometry, Control

Journal: Cell death & disease

Article Title: Therapy-induced senescence of glioblastoma cells is determined by the p21 CIP1 -CDK1/2 axis and does not require activation of DREAM.

doi: 10.1038/s41419-025-07651-8

Figure Lengend Snippet: Fig. 2 Analysis of DREAM activation. A LN229 cells were exposed to 50 µM TMZ for 96 or 144 h. Expression and phosphorylation of p130 (pp130), Rb (pRb), as well as expression of p21CIP1, E2F1, E2F4, E2F5, B-Myb, FOXM1, LIN9, and p27 were measured by immunoblotting. β-Actin or HSP90 was used as internal loading control. Quantification of the immunoblot indicates x-fold induction in TMZ-exposed cells compared to untreated cells. B, C LN229 cells were exposed to 50 µM TMZ for 48 or 144 h. B Expression of FOXM1, MYBL2, CDK1, CDKN1A, CDKN1B, E2F1, E2F4 and E2F5 was measured by qPCR. C Expression of CCNA1, CCNA2, CCNB1, CCNB2, CCND1, CCND2, CCNE1 and CCNE2 was measured by qPCR. B, C Experiments were performed in triplicates, ACTB and GAPDH were used as internal loading control. Differences between the control and TMZ treatment were statistically analyzed using Student’s t test (non-labeled = not significant; *p < 0.1; **p < 0.01; ***p < 0.001).

Article Snippet: For gene silencing, predesigned siRNAs specific for p21CIP1 (sc-29427, Santa Cruz Biotechnology) were used; control human non-silencing siRNA (Silencer Select Predesigned siRNA Negative Control #1 siRNA; Ambion) was used as negative control.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Western Blot, Control, Labeling

Journal: Cell death & disease

Article Title: Therapy-induced senescence of glioblastoma cells is determined by the p21 CIP1 -CDK1/2 axis and does not require activation of DREAM.

doi: 10.1038/s41419-025-07651-8

Figure Lengend Snippet: Fig. 4 Binding of p21CIP1 to CDKs and activation of the DDR. A LN229 cells were exposed to 50 µM TMZ for 48 or 144 h. Interaction between p21CIP1 and CDK1, CDK2, and CDK4 was measured by co-immunoprecipitation. B LN229 cells were exposed to 50 µM TMZ for 48, 96, or 144 h. Expression of CDK1, CDK2, and CDK4 was measured by immunoblotting. β-Actin was used as internal loading control. C LN229 cells were exposed to 50 µM TMZ for 24, 48, or 72 h. D LN229 cells were exposed to 50 µM TMZ for 96 or 144 h. C, D Expression of p21CIP1, CDC25c and PLK1, as well as expression and phosphorylation of CDK1 (pCDK1), CHK1 (pCHK1), WEE1 (pWEE1) and B-Myb (pB-Myb) was measured by immunoblotting. β-Actin or HSP90 were used as internal loading control. B, C Quantification of the immunoblot indicates x-fold induction in TMZ exposed cells compared to untreated cells.

Article Snippet: For gene silencing, predesigned siRNAs specific for p21CIP1 (sc-29427, Santa Cruz Biotechnology) were used; control human non-silencing siRNA (Silencer Select Predesigned siRNA Negative Control #1 siRNA; Ambion) was used as negative control.

Techniques: Binding Assay, Activation Assay, Immunoprecipitation, Expressing, Western Blot, Control, Phospho-proteomics

Journal: Cell death & disease

Article Title: Therapy-induced senescence of glioblastoma cells is determined by the p21 CIP1 -CDK1/2 axis and does not require activation of DREAM.

doi: 10.1038/s41419-025-07651-8

Figure Lengend Snippet: Fig. 6 Binding of p21CIP1 to CDKs and activation of the DDR in A172 and U87MG cells. A U87MG and A172 cells were exposed to 50 µM TMZ for 96 or 144 h. Expression of p21CIP1, CDC25c, and PLK1, as well as expression and phosphorylation of CDK1 (pCDK1), CHK1 (pCHK1), WEE1 (pWEE1) and B-Myb (pB-Myb) were measured by immunoblotting. β-Actin or HSP90 was used as an internal loading control. Quantification of the immunoblot indicates x-fold induction in TMZ-exposed cells compared to untreated cells. B U87MG and A172 cells were exposed to 50 µM TMZ for 48 or 144 h. Interaction between p21CIP1 and CDK1, CDK2, and CDK4 was measured by co-immunoprecipitation. C LN229, U87MG, and A172 cells were exposed to 50 µM TMZ for 48 or 144 h. In addition, the cells were exposed to 1 µM Palbociclib for 144 h or the cells were exposed to 50 µM TMZ, and 48 h later 1 µM Palbociclib was added for additional 96 h (TMZ/Palbo). Cell death and cell cycle distribution were measured by flow cytometry using PI staining; experiments were performed in triplicates. Differences between the frequency of cells in the G2-phase or showing a DNA content >2n after TMZ and TMZ/Palbo treatment were statistically analyzed using Student’s t test (***p < 0.001).

Article Snippet: For gene silencing, predesigned siRNAs specific for p21CIP1 (sc-29427, Santa Cruz Biotechnology) were used; control human non-silencing siRNA (Silencer Select Predesigned siRNA Negative Control #1 siRNA; Ambion) was used as negative control.

Techniques: Binding Assay, Activation Assay, Expressing, Phospho-proteomics, Western Blot, Control, Immunoprecipitation, Cytometry, Staining