p21 primary antibody waf1/cip1/cdkn1a p21 sc-6246  (Santa Cruz Biotechnology)

Journal: Scientific Reports

Article Title: Vibrational spectroscopy unveils distinct cell cycle features of cancer stem cells in melanoma

doi: 10.1038/s41598-025-14018-8

Figure Lengend Snippet: Immunohistochemical staining of p16 and p21 expression in CHL-1 melanoma cells, CD133 + melanoma stem-like cells, and CD133- non-stem melanoma cells at different time points (11 h, 24 h, 48 h, and 72 h). Representative images show the time-dependent expression of p16 and p21 in each cell type, with higher magnification insets. Scale bars: 100 μm. Bar graphs on the right represent the quantification of p16- and p21-positive cells (mean ± SD) for each group at the indicated time points.

Article Snippet: Primary antibodies against p21 (Bioss, BB07142233) and p16 (Bioss, BB10114929) were diluted at 1:200 and applied to the cells.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cell Death Discovery

Article Title: Bisphenol-A disrupts mitochondrial functionality leading to senescence and apoptosis in human amniotic mesenchymal stromal cells

doi: 10.1038/s41420-025-02620-8

Figure Lengend Snippet: Expression of p53, p21 and p27 cell-cycle regulating genes was analysed by RT-PCR 24 h after exposure to increasing BPA concentrations (0.05, 0.1, 0.2, 0.3, 0.35 and 0.4 μM). Results are presented as fold-change relative to control conditions (MetOH) ( A ). Furthermore, p21 protein expression and its nuclear translocation in hAMSC were assessed 24 h after exposure to increasing BPA concentrations (0.1, 0.2, 0.3, and 0.4 μM) using immunofluorescence analysis. Pictures were acquired at ×20 magnification (scale bar, 50 μm) ( B ). p21-positive cells were identified by a rosy-red signal, while nuclei were stained with DAPI (blue). The total number of p21 positive cells was quantified and is reported in ( C ). Fluorescence intensity of p21, measured as Normalized Integrated Density, is presented in ( D ). Histograms represent the mean values ± standard deviation from n = 4 ( A ) and n = 3 ( B ) independent experiments. Statistical analysis was performed versus the control condition represented by the MetOH: p < 0.01(**), p < 0.001(***), p < 0.0001(****).

Article Snippet: Subsequently, cells were washed three times with Tris Buffered Saline (TBS) for 5 min each. hAMSC were incubated with p21 primary antibody (Waf1/Cip1/CDKN1A p21, sc-6246, Santa Cruz Biotechnology, Texas, USA), diluted 1:100 in normal goat serum (NGS, Invitrogen, Waltham USA, #10000C) and incubated overnight at 4 °C in the dark.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Standard Deviation

Journal: Cell Death Discovery

Article Title: Bisphenol-A disrupts mitochondrial functionality leading to senescence and apoptosis in human amniotic mesenchymal stromal cells

doi: 10.1038/s41420-025-02620-8

Figure Lengend Snippet: Alteration in mitochondrial function, evidenced by enhanced production of ROS in hAMSC after BPA exposure, trigger the downstream activation of several signalling pathways. ROS accumulation activates an antioxidant response, which is marked by an elevated production of Nrf2 and HO-1. Concurrently, the high ROS level induce sterile inflammation, resulting in increased transcription of factors involved in inflammasome complex formation and activation. However, this increased transcription does not translate to higher production of IL-1β, the downstream effector of the inflammasome pathway. Instead, oxidative stress promotes p53 stabilization and upregulates p21 and p27 genes, as well as components of the senescence-associated secretory phenotype (SASP). Ultimately, the senescent state serves as a prelude to apoptosis, which occurs when hAMSC are exposed to the highest BPA concentrations.

Article Snippet: Subsequently, cells were washed three times with Tris Buffered Saline (TBS) for 5 min each. hAMSC were incubated with p21 primary antibody (Waf1/Cip1/CDKN1A p21, sc-6246, Santa Cruz Biotechnology, Texas, USA), diluted 1:100 in normal goat serum (NGS, Invitrogen, Waltham USA, #10000C) and incubated overnight at 4 °C in the dark.

Techniques: Activation Assay, Sterility

Journal: Molecular Medicine Reports

Article Title: Natural bioactive gallic acid shows potential anticancer effects by inhibiting the proliferation and invasiveness behavior in human embryonic carcinoma cells

doi: 10.3892/mmr.2025.13516

Figure Lengend Snippet: Primer sequences for reverse transcription-quantitative PCR.

Article Snippet: The primary antibodies were purchased from Santa Cruz Biotechnology, Inc.: p21 (cat. no. sc-756), p53 (cat. no. sc-126), cyclin E (cat. no. sc-481), MMP9 (cat. no. sc-13520), STAT5b (cat. no. sc-1656), CDK4 (cat. no. sc-260) and β-actin (cat. no. sc-47778).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Natural bioactive gallic acid shows potential anticancer effects by inhibiting the proliferation and invasiveness behavior in human embryonic carcinoma cells

doi: 10.3892/mmr.2025.13516

Figure Lengend Snippet: GA enhances the arrest of G 0 /G 1 cell cycle in CSCs. (A) The flow cytometry results demonstrated the cell cycle distribution with 0 or 200 µM GA, respectively, for 24 h. Graphics display a G 0 /G 1 arrest by GA in embryonic CSCs. (B) Immunoblotting analysis showed the expression patterns of p21, p27, p53, CDK4 or cyclin D1 and E with 0, 100, or 200 µM GA, respectively, for 24 h. All proteins were normalized to β-actin levels. (C) Reverse transcription-quantitative PCR represented the genetic expression of cell cycle factors, including CDKN1A, CDKN1B, CDK4, CCND1 or CCNE1 mRNA. ***P<0.001 (ANOVA test) and # P<0.001 vs. control. GA, gallic acid; CSCs, cancer stem cells.

Article Snippet: The primary antibodies were purchased from Santa Cruz Biotechnology, Inc.: p21 (cat. no. sc-756), p53 (cat. no. sc-126), cyclin E (cat. no. sc-481), MMP9 (cat. no. sc-13520), STAT5b (cat. no. sc-1656), CDK4 (cat. no. sc-260) and β-actin (cat. no. sc-47778).

Techniques: Flow Cytometry, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Cancer

Article Title: Therapy-induced senescence is a transient drug resistance mechanism in breast cancer

doi: 10.1186/s12943-025-02310-0

Figure Lengend Snippet: Chemotherapy-surviving cells transition into a transient senescent state. A Representative growth kinetics of cell cultures following 5-day doxorubicin (DOX) treatment. DOX was administered on day 0, and the medium was refreshed on day 5. The minimum cell counts are highlighted on the curves (red). B X-Gal staining of control (CTR), therapy-induced senescent (TIS), and repopulated (REPOP) cells, accompanied by quantification of staining intensity. C Western blot analysis of senescence marker CDKN1A and LMNB1 protein expression in CTR, TIS, and REPOP cells, with quantification of relative protein levels. D Fluorescence microscopy detection of DNA damage (γ-H2AX), senescence-associated β-galactosidase (SA-β-Gal) activity, mitochondria, lysosomes, and nucleoli in CTR and TIS cells. Scale bar: 20 µm

Article Snippet: The membranes were incubated with CDKN1A (p21), LMNB1 or Lamin B1 (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (Thermo Fisher, Waltham, MA, USA) and Bcl-XL (Proteintech, Rosemont, USA) primary antibodies at 4°C overnight and then incubated with HRP-conjugated secondary antibodies (List of all antibodies used in this study can be found in the Supplementary Material 1).

Techniques: Staining, Control, Western Blot, Marker, Expressing, Fluorescence, Microscopy, Activity Assay

Journal: Molecular Cancer

Article Title: Therapy-induced senescence is a transient drug resistance mechanism in breast cancer

doi: 10.1186/s12943-025-02310-0

Figure Lengend Snippet: Therapy-induced senescence (TIS) cells that escape senescence drive repopulation after chemotherapy. A UMAP projection of single-cell transcriptomes from the MCF7 breast cancer cell line, illustrating distinct clustering of control (CTR, dark brown), therapy-induced senescent (TIS, scarlet), and repopulating (REPOP, light brown) cell populations. The separation of these clusters indicates transcriptionally distinct states, with TIS cells forming a well-defined cluster distinct from CTR and REPOP populations. The REPOP population shows a partial transcriptional shift toward CTR, reflecting its reversal from the TIS state. B UMAP projection of single-cell transcriptomes from the T47D breast cancer cell line, similarly showing distinct clustering of CTR (dark green), TIS (crimson), and REPOP (light green) cell populations. The clustering pattern resembles that observed in MCF7 cells, with a well-separated TIS population and REPOP cells positioned between TIS and CTR clusters, suggesting partial transcriptional reversion. C Integrated UMAP analysis of MCF7 and T47D cell lines, combining data from both models to highlight cell type-specific transcriptomic profiles. Control populations (MCF7 CTR in dark brown; T47D CTR in dark green) form distinct clusters, whereas TIS populations from both cell lines (scarlet for MCF7; crimson for T47D) also exhibit clear separation from their respective CTR counterparts. The integration further reveals that despite the transcriptional similarities in senescence-associated programs, MCF7 and T47D maintain cell-line-specific transcriptomic differences, as reflected in their segregated distributions. D Feature plots showing the expression of key senescence and proliferation-related markers in individual MCF7 cells. CDKN1A (p21) is upregulated in TIS cells (top left panel), confirming cell cycle arrest. Conversely, markers associated with proliferation, including LMNB1, TOP2A, and MKI67, are downregulated (top right and bottom panels), consistent with the senescent phenotype. The presence of scattered high-expressing cells within the TIS population suggests the existence of ‘escaper’ subpopulations that may retain some proliferative capacity. E Feature plots of CDKN1A, LMNB1, TOP2A, and MKI67 expression in T47D cells, showing a similar transcriptional profile to MCF7 cells. CDKN1A is significantly upregulated in TIS cells, while LMNB1, TOP2A, and MKI67 are markedly downregulated. As in MCF7, a fraction of TIS cells display non-uniform expression of these markers, indicating potential escape from the senescent state. F UMAP projection of MCF7 cells overlaid with cell cycle phase information (G1: green; S: blue; G2/M: red). Pie charts illustrate the proportional distribution of cells in each phase across the CTR, TIS, and REPOP populations. TIS cells predominantly reside in the G1 phase (79.7%), reflecting irreversible cell cycle arrest, whereas REPOP cells show an increased proportion of cycling cells, particularly in the G2/M phase, indicating their proliferative re-entry. G UMAP projection of T47D cells colored by cell cycle phases, with corresponding pie charts depicting phase distributions in CTR, TIS, and REPOP populations. TIS cells in T47D exhibit a similar G1 arrest phenotype as observed in MCF7, while REPOP cells regain a more balanced cell cycle distribution, mirroring their recovery from senescence. H Gene set enrichment analysis (GSEA) of differentially expressed genes in MCF7 TIS cells compared to CTR, highlighting significant enrichment of senescence-associated pathways (e.g., inflammatory response, DNA damage signaling) and suppression of proliferation-associated pathways. Normalized enrichment scores (NES) are shown, with positive values indicating upregulated pathways and negative values indicating suppressed pathways. I GSEA results for T47D TIS cells, demonstrating pathway-level alterations similar to those observed in MCF7, with enrichment of senescence-associated programs and suppression of cell cycle progression. J Schematic representation of the senescence induction and reversion process. Cells undergo therapy-induced senescence (TIS) following exposure to doxorubicin (DOX). Over time, a subset of TIS cells escape growth arrest and re-enter the cell cycle, forming the REPOP population. This process involves transcriptional reprogramming, with a balance between senescent, proliferating, and apoptotic fates. K UMAP trajectory analysis of MCF7 cells depicting the transition from CTR to TIS (bottom panel) and from TIS to REPOP (top panel). Cells are colored based on pseudotime, capturing the progressive shift in transcriptional states. TIS cells form a distinct branch, while REPOP cells demonstrate convergence toward CTR, reflecting their transcriptional plasticity. L UMAP trajectory analysis of T47D cells, analogous to MCF7. The bottom panel illustrates the transition from CTR to TIS, while the top panel depicts the transition from TIS to REPOP. The REPOP population becomes almost identical to the CTR cluster, exhibiting highly overlapping transcriptional profiles, even more profoundly than in MCF7. This supports a model of senescence escape and complete proliferative recovery. M Heatmap of gene expression changes in a curated set of genes associated with drug resistance, senescence regulation, and cell cycle control across CTR, TIS, and REPOP states in both MCF7 and T47D cell lines. Genes exhibit dynamic expression patterns, with key senescence markers upregulated in TIS and downregulated upon REPOP transition, while drug resistance-associated genes show variable trends between cell lines. This highlights the complex interplay between senescence, proliferation, and therapy resistance

Article Snippet: The membranes were incubated with CDKN1A (p21), LMNB1 or Lamin B1 (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (Thermo Fisher, Waltham, MA, USA) and Bcl-XL (Proteintech, Rosemont, USA) primary antibodies at 4°C overnight and then incubated with HRP-conjugated secondary antibodies (List of all antibodies used in this study can be found in the Supplementary Material 1).

Techniques: Control, Expressing, Gene Expression