Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a IB analysis of WCLs derived from HEK293T cells transfected with His-E4F1 and increasing doses of Flag-PA28γ. b Quantitative real-time PCR analysis of the indicated genes in HEK293T cells transfected with EV and increasing doses of Flag-PA28γ. Endo., endogenous. c HSC-3 cells stably expressing EV or Flag-PA28γ were treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of E4F1 levels relative to tubulin levels is shown. d , e HEK293T cells stably expressing EV or Flag-PA28γ were transfected with Myc-E4F1 truncations, and treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of Myc-E4F1 levels relative to tubulin levels is shown. f HEK293T cells transfected with the indicated plasmids were treated without or with 10 μM MG132 for 6 h before harvesting, and WCLs were collected for IB analysis. g HEK293T cells stably expressing shRNA against endogenous PA28γ were transfected with the indicated plasmids, and WCLs were collected for IB analysis. h Purified PA28γ, E4F1 and 20S proteasome in the absence or presence of 100 nM proteasome inhibitor epoxomicin (Epox) were incubated as indicated for 45 min, followed by IB analysis. i Purified PA28γ WT, PA28γ-T23A, E4F1, p21 (as a positive control for E4F1) and 20S proteasome were incubated as indicated for 45 min, followed by IB analysis. Data in ( b – e ) represent the mean ± SD of three biological replicates; statistical significance was assessed by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human E4F1 protein (H00001877-P01) and recombinant human p21/CDKN1A protein (NBP2-22976) were purchased from Novus Biologicals.

Techniques: Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, shRNA, Purification, Incubation, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide-Activated Macrophages Suppress Cellular Senescence and Promote Rejuvenation in Human Dermal Fibroblasts

doi: 10.3390/ijms26157061

Figure Lengend Snippet: Expression of the p16 and p21 genes in Young cells. The schematic protocol (1)–(7) is shown at the top of the figure. The test conditions in figures ( A – D ) correspond to the schematic protocol (1)–(7). Young cells were cultured for 24 h after replacing half of the medium with EMEM, Old cell-conditioned medium (Old CM), or THP/LPS-conditioned medium (THP/LPS-CM; 0–1000 ng/mL). ( A , B ) Total RNA was extracted, and the relative mRNA expression levels of p16 ( A ) and p21 ( B ) were measured by Quantitative real-time PCR (RT-qPCR) using GAPDH as a housekeeping gene. ( C , D ) Young cells were treated with EMEM or THP/LPS-CM (0, 10, or 100 ng/mL) for 24 h. Cells were lysed, and 12.5 μg of total protein per lane was separated by SDS-PAGE, followed by western blot analysis using antibodies against P16 ( C ) or P21 ( D ). Representative western blot images are shown in the boxed regions of panels C and D. Band intensities were normalized to β-actin and quantified. Data are presented as mean ± SD (n = 3). Different letters (a–c) indicate statistically significant differences ( p < 0.05, ANOVA, Tukey–Kramer).

Article Snippet: Proteins were resolved via 12.5% SDS-PAGE at 30 mA for 90 min and transferred onto PVDF membranes at 300 mA for 1 h. Membranes were then blocked with PBS containing 0.1% Tween-20 and 5% skim milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies against P21 Waf1/Cip1 (1:1000) and β-actin (1:2000; both from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide-Activated Macrophages Suppress Cellular Senescence and Promote Rejuvenation in Human Dermal Fibroblasts

doi: 10.3390/ijms26157061

Figure Lengend Snippet: Changes in P16, P21, and Ki-67 expression in senescent NB1RGB cells treated with THP/LPS-CM. The schematic protocol (1)–(7) is shown at the top of the figure. The test conditions in figures ( A – D ) correspond to the schematic protocol (1)–(7). ( A ) Young and Old NB1RGB cells were treated with EMEM, THP/LPS (0 ng/mL)-CM, or THP/LPS (100 ng/mL)-CM for 24 h. Relative mRNA expression levels of p16 , p21 , and Ki-67 were analyzed by RT-qPCR and normalized to the levels in Young cells. ( B ) Old cells were treated with LPS (10, 100, or 1000 ng/mL) for 24 h, and the expression levels of p16 , p21 , and Ki-67 were assessed by RT-qPCR. In both ( A , B ), bars represent mean ± SD (n = 5). Different letters (a–c) indicate statistically significant differences ( p < 0.05). ( C ) Young and Old cells were treated with EMEM, THP/LPS (0 ng/mL)-CM, or THP/LPS (100 ng/mL)-CM for 24 h, then immunofluorescence staining was performed using antibodies against P21 and Ki-67 (n = 5). Representative fluorescence images were acquired using a 20× objective lens with 2× digital zoom. In P21 and Ki-67 single-staining panels green = P21 or Ki-67, red = actin, and blue = nuclei. In double-staining panels green = P21, red = Ki-67, and blue = nuclei. Scale bars = 50 μm. ( D ) Based on the immunofluorescence images, the percentages of P21-positive cells, Ki-67-positive cells, and total cell counts per field were quantified. Data are expressed as mean ± SD (n = 5). Different letters (a–c) indicate statistically significant differences ( p < 0.05, ANOVA, Tukey–Kramer).

Article Snippet: Proteins were resolved via 12.5% SDS-PAGE at 30 mA for 90 min and transferred onto PVDF membranes at 300 mA for 1 h. Membranes were then blocked with PBS containing 0.1% Tween-20 and 5% skim milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies against P21 Waf1/Cip1 (1:1000) and β-actin (1:2000; both from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence, Double Staining

Journal: Antioxidants

Article Title: Epicatechin Gallate Ameliorates UVB-Induced Photoaging by Inhibiting p38α-Mediated Autophagy and Oxidative Stress

doi: 10.3390/antiox15020180

Figure Lengend Snippet: Effects of ECG on UVB-induced senescence markers in HaCaT cells. ( A , B ) Cell cycle distribution analysis of HaCaT cells by flow cytometry. The histogram in ( A ) shows DNA content (propidium iodide staining intensity) on the x-axis and cell count on the y-axis. The stacked bar chart in ( B ) quantifies the percentage of cells in different cell cycle phases (G0/G1, S, and G2/M). ( C ) Relative mRNA expression levels of p53 , p16 , p21 , IL6 , and TNF-α determined by RT-qPCR. ( D , E ) Representative Western blots and densitometric analysis of p53, p16, and p21 proteins. ( F , I ) Representative immunofluorescence images of γH2AX and Lamin B1 (scale bar: 50 μm). γH2AX foci are shown in green, Lamin B1 is shown in red, and nuclei are counterstained with DAPI (blue). White arrows indicate nuclei with damage. ( G ) Representative images of SA-β-gal staining in HaCaT cells (scale bar: 200 μm). ( H ) Quantitative analysis of SA-β-gal positive cells. Data are presented as the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Rabbit anti-Beclin1 (BECN1) antibody (cat. #11306), mouse anti-tubulin antibody (cat. #66031-1-IG), rabbit anti-LaminB1 (LMNB1) antibody (cat. #12987-1-AP), rabbit anti-Cyclin-Dependent Kinase Inhibitor 1A (p21, CDKN1A) antibody (cat. #10355-1-AP) and rabbit anti-LC3 antibody (cat. #14600) were purchased from Proteintech group (Wuhan, China).

Techniques: Flow Cytometry, Staining, Cell Characterization, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Science advances

Article Title: Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

doi: 10.1126/sciadv.adh1436

Figure Lengend Snippet: Fig. 2. Dual MDM2/XPO1 inhibition enhances p53 target transcription and dysregulates MYC transcriptional program, leading to cell cycle arrest. (A) Relative quantitation (RQ) values by quantitative PCR for CDKN1A and MDM2 in OCI-AML3 cells treated with Mil, Sel, or Mil + Sel for the indicated time points. (B) Pathway analyses comparing Mil versus Mil + Sel and Sel versus Mil + Sel in RNA-seq in OCI-AML3 cells treated with Mil, Sel, or Mil + Sel for 12 hours. The top up-regulated and down- regulated pathways are indicated by red and blue arrows, respectively. NES, normalized enrichment score. (C) Volcano plots [beta score (magnitude) and q values (sig- nificance, −log10 scale)] from differential gene expression profiles in RNA-seq from OCI-AML3 cells DMSO versus Mil (left), Sel (middle), and versus Mil + Sel (right). The top 10 up-regulated TP53 targets and down-regulated MYC targets are indicated with red and blue colors, respectively. The remaining genes are indicated in gray. (D) Cell cycle analyses using multiparameter flow cytometry with Ki-67, p21, and cleaved caspase-3 in OCI-AML3 cells treated with Mil, Sel, or Mil + Sel for 24 hours. Caspase-3– negative cells were gated for EdU, Ki-67, and p21 panels. (E and F) Change in the percentages of cells in S (E) and M (F) phases in OCI-AML3 cells in indicated treatments. (G) The percentages of “p21 high” OCI-AML3 cells [rectangles shown in the third row of (D)] in indicated treatments. (H) Change of cell percentages of cleaved caspase-3– positive OCI-AML3 cells [rectangles shown in the fourth row of (D)] in indicated treatments for G1 (2N) and G2-M (4N) phases. A Mil and Sel concentration of 160 nM was used. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Antibodies used were those against Ki-67 (PE-Cy7; BD Biosciences, #561283), cleaved caspase-3 [fluorescein isothiocyanate (FITC); BD Biosciences, #559341], and p21 (AF700, R&D Systems, Minneapolis, MN, USA, #IC1047N).

Techniques: Inhibition, Quantitation Assay, Real-time Polymerase Chain Reaction, RNA Sequencing, Gene Expression, Flow Cytometry, Concentration Assay

Journal: Science advances

Article Title: Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

doi: 10.1126/sciadv.adh1436

Figure Lengend Snippet: Fig. 6. Triple inhibition of MDM2, XPO1, and BCL2 induces apoptosis in p53 reactivation–responsive persistent AML cells with increased MCL1 along with stress responses. (A) t-distributed stochastic neighbor embedding (t-SNE) plots generated by unsupervised clustering and dimension reduction using RphenoGraph. Clusters are annotated by numbers with distinct colors. (B) The t-SNE plot shown in (A) along with marker expression plots by t-SNE for CD34, p53, p21, MCL1, LC3B, and ATF4. Cluster 4 is connected by red lines and rectangles. (C) t-SNE plots of samples treated with each indicated treatment. Clusters 11, 12, and 13; cluster 4; and cluster 8 are circled by black, red, and orange dashed lines, respectively. (D) Proportions of each cluster in each sample with the indicated treatment in 195Pt-negative live cells. The colors correspond with the ones in the t-SNE plot shown in (A). (E) Immunoblots of indicated proteins collected from OCI-AML3 cells with indicated treatments (100 nM of Mil, Sel, and/or Ven for 24 hours).

Article Snippet: Antibodies used were those against Ki-67 (PE-Cy7; BD Biosciences, #561283), cleaved caspase-3 [fluorescein isothiocyanate (FITC); BD Biosciences, #559341], and p21 (AF700, R&D Systems, Minneapolis, MN, USA, #IC1047N).

Techniques: Inhibition, Generated, Marker, Expressing, Western Blot

Journal: eLife

Article Title: Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

doi: 10.7554/eLife.70691

Figure Lengend Snippet: ( A ) NHF-1 cells were engineered to express a TET-inducible HA-p130(WT) or HA-p130(AA) transgene. Doxycycline (100 ng/ml) was used to induce p130 expression, and water was used as a vehicle control. HA-p130 levels were assessed by immunoblot after cells were grown in doxycycline for up to 14 days. ( B ) Inducible p130 NHF-1 cells were grown in 100 ng/ml doxycycline-containing media for 14 days to induce p130 expression. Cells were counted on the indicated days. Data represent mean ± SEM for n=3 independent experiments. ( C ) Doubling time was calculated from counting experiment in ( B ). Error bars are SEM for n=3 independent experiments. Representative of n=2 independent experiments. ( D ) NHF-1 cells were grown in media supplemented with nocodazole for 16 hr, then immunoblotted for the indicated proteins. Representative of n=2 independent experiments. ( E–G ) Inducible p130 NHF-1 cells were grown in 100 ng/ml doxycycline for indicated times. Cells were fixed and analyzed by iterative immunofluorescent staining and imaging. Distributions of single-cell measurements are shown for nuclear phosphorylated versus total RB ( E , left) and representative images of phosphorylated RB are shown for indicated days ( E , right). Distributions of single-cell measurements are also shown for cytoplasmic p21 ( F ) and nuclear versus cytoplasmic p130 ( G ), as indicated.

Article Snippet: Sample was incubated with 4i blocking solution (sBS: 100 mM maleimide [Sigma-Aldrich, 129585], 100 mM NH 4 Cl [Sigma-Aldrich, A9434], and 1% bovine serum albumin in PBS) for 1 hr and incubated with primary antibodies diluted as required (anti-phospho-RB [S807/S811] [1:1000, Cell Signaling Technology, 8516]; anti-RB [1:500, Cell Signaling Technology, 9309]; anti-p21 [1:200, R&D Systems, AF1047]; anti-p130 [1:100, Cell Signaling Technology, 13610]; anti-phospho-H2A.X [Ser139] [1:200, Cell Signaling Technology, 80312], anti-phospho-CHK1 [S317] [1:800, Cell Signaling Technology, 12302], anti-53BP1 [1:250, Abcam, ab36823], CDT1 [1:200, Cell Signaling Technology, 8064], CDC6 [1:100, Santa Cruz, sc-9964], HA [1:200, BioLegend, 901501]) in conventional blocking solution (cBS: 1% bovine serum albumin in PBS) overnight at 4°C.

Techniques: Expressing, Control, Western Blot, Staining, Imaging

Journal: eLife

Article Title: Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

doi: 10.7554/eLife.70691

Figure Lengend Snippet:

Article Snippet: Sample was incubated with 4i blocking solution (sBS: 100 mM maleimide [Sigma-Aldrich, 129585], 100 mM NH 4 Cl [Sigma-Aldrich, A9434], and 1% bovine serum albumin in PBS) for 1 hr and incubated with primary antibodies diluted as required (anti-phospho-RB [S807/S811] [1:1000, Cell Signaling Technology, 8516]; anti-RB [1:500, Cell Signaling Technology, 9309]; anti-p21 [1:200, R&D Systems, AF1047]; anti-p130 [1:100, Cell Signaling Technology, 13610]; anti-phospho-H2A.X [Ser139] [1:200, Cell Signaling Technology, 80312], anti-phospho-CHK1 [S317] [1:800, Cell Signaling Technology, 12302], anti-53BP1 [1:250, Abcam, ab36823], CDT1 [1:200, Cell Signaling Technology, 8064], CDC6 [1:100, Santa Cruz, sc-9964], HA [1:200, BioLegend, 901501]) in conventional blocking solution (cBS: 1% bovine serum albumin in PBS) overnight at 4°C.

Techniques: Plasmid Preparation, Mutagenesis, Flow Cytometry, SYBR Green Assay, Western Blot, Transfection, Construct, Expressing, Sequencing, Subcloning, Cytometry

Journal: Nutrients

Article Title: Malva sylvestris Flower Extract Exhibits Antineoplastic Potential Against Human Colon Cancer Cell Lines and Induces CDK2 Transcript Instability via Plant miR160-5p

doi: 10.3390/nu18030495

Figure Lengend Snippet: Molecular mechanisms activated by MFE in the tumour cells. ( A – D ) Results of RT-qPCR analyses carried out on the RNA isolated from HCT-116 (panels ( A , C )) and Caco-2 (panels ( B , D )) cells treated for 48 h with the highest dose of MFE. The data relative to VIM , E-CAD , ITGB3 (panels ( A , B )), P53 , P27 and P21 (panels ( C , D )) transcript levels were expressed as fold change compared to the respective internal CNT taken as unit (i.e., 1), after normalization for β-ACTIN mRNA according to the 2 −∆∆Ct formula. ( E – G ) Representative immunoblots of P53, P27, P21 (panel ( E )), phospho c-MYC (Ser62), phospho CDK2 (Thr160), phospho AKT (Ser 473), AKT and β-ACTIN (panel ( F )) protein levels in HCT-116 and Caco-2 cells, exposed or not for 48 h to the highest dose of MFE. Considering the protein signal of the CNT as a unit (i.e., 1), the quantitation of each band is reported immediately below its representative image in the panel ( E ). Results of the immunoblots presented in panel ( F ) are reported in the form of graphs in panel ( G ) as percentage variation compared to the CNT taken as unit (100%). β-ACTIN was used as a loading control to normalise the results. ( H ) The graph shows the transfection efficiency of the synthetic miR160b-5p after transfection in HCT-116 and Caco-2 cells for 48 h. ( I ) CDK2 mRNA levels, measured by RT-qPCR, in HCT-116 and Caco-2 cells transfected for 48 h with synthetic miR160b-5p. Data were reported relative to CNT cells transfected with a scramble miRNA (SiR) and taken as unit (i.e., 1) after normalization for β-ACTIN mRNA, according to the 2 −∆∆Ct formula. ( J ) Representative immunoblots of phospho c-MYC (Ser62), phospho CDK2 (Thr160) and β-ACTIN protein levels in HCT-116 and Caco-2 cells transfected for 48 h with synthetic miR160b-5p. Considering the protein signal of the CNT as a unit (i.e., 1), the quantitation of each band was reported immediately below its representative image. β-ACTIN was used as a loading control for the normalisation. In all panels, data were indicated as mean ± s.d. of three independent experiments (* p < 0.05 vs. control; ** p < 0.01; *** p < 0.001).

Article Snippet: Rabbit polyclonal phoshpo-AKT (Ser473) (cs-4060), mouse monoclonal cleaved CASPASE-3 (cCASP-3) (cs-94530), rabbit monoclonal phospho-CDK2 (Thr160) (cs-2561), mouse monoclonal phosho-c-MYC (Ser62) (cs-13748) and mouse monoclonal P21 WAF1/Cip1 (cs-2946) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Quantitative RT-PCR, Isolation, Western Blot, Quantitation Assay, Control, Transfection

Journal: Nutrients

Article Title: Malva sylvestris Flower Extract Exhibits Antineoplastic Potential Against Human Colon Cancer Cell Lines and Induces CDK2 Transcript Instability via Plant miR160-5p

doi: 10.3390/nu18030495

Figure Lengend Snippet: Hypothetical model of MFE action in tumour cells. The possible signalling activated in HCT-116 (on the left) and Caco-2 (on the right) cells treated with MFE (indicated with light violet spots) is shown. In the HCT-116 cells, a block of senescence mediated by an upregulation of the CDK2/c-MYC/AKT axis and a hypothetic differentiation event modulated by cytoplasmatic P27 is outlined. On the other hand, in Caco-2 cells, the activation of senescence mediated by an increase in P21, a reduction in AKT and an accumulation of intracellular ROS levels is represented. Legend—green up arrows indicate an increase in the related components inside the cell; red down arrows indicate a decrease in the related components inside the cell; continuous black arrows indicate that the signal is active; dashed black arrows indicate that the signal is not active; black arrows with tips indicate a promoting activity; black arrows without tips indicate inhibitory activity. Abbreviations—Cyt: cytoplasm; CM: cell membrane; ES: extracellular space; Nuc: nucleus; P: phosphorylated protein; ROS: reactive oxygen species.

Article Snippet: Rabbit polyclonal phoshpo-AKT (Ser473) (cs-4060), mouse monoclonal cleaved CASPASE-3 (cCASP-3) (cs-94530), rabbit monoclonal phospho-CDK2 (Thr160) (cs-2561), mouse monoclonal phosho-c-MYC (Ser62) (cs-13748) and mouse monoclonal P21 WAF1/Cip1 (cs-2946) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Blocking Assay, Activation Assay, Activity Assay, Membrane

Journal: Inflammation research : official journal of the European Histamine Research Society ... [et al.]

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype.

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: Fig. 3 NLRP1 contributes to cellular senescence in vivo. A Nlrp1 protein expression in liver from WT mice at 1 month after IR. B Serum levels of IL-18 after IR. C Effect of IR on the bodyweight of WT and Nlrp1 knockout (KO) mice. D Protein expression in liver from IR and non-IR WT and Nlrp1 KO mice of senescent mark- ers (IL-6, p16 and p21). Densitometry in Supplementary Fig. 6. E Serum levels of IL-6 in serum from IR and non-IR WT and Nlrp1 KO mice. F Heat map depicting expression of 44 mouse cytokines in serum at 5 weeks after IR of WT and Nlrp1 KO mice. n = 6 mice

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP229463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: In Vivo, Expressing, Knock-Out

Journal: International journal of molecular sciences

Article Title: Obacunone Photoprotective Effects against Solar-Simulated Radiation-Induced Molecular Modifications in Primary Keratinocytes and Full-Thickness Human Skin.

doi: 10.3390/ijms241411484

Figure Lengend Snippet: Figure 1. Damaging effects of solar-simulated radiation on a reconstructed full-thickness (FT) human skin. FT skin models were irradiated with 4 J/cm2, 12 J/cm2, and 20 J/cm2 of solar-simulated radiation (SSR). (A) LDH cytotoxicity assay. (B) Paraffin tissue sections were stained with hematoxylin and eosin. Black arrows indicate localization of vacuolization, and red arrows indicate photodamaged keratinocytes (sunburn cells). Scale bar: 100 µM. (C–E) IL-1α, IL-6, and IL-8 levels were measured by ELISA. (F,G) p21 and p53 mRNA levels were measured by real-time PCR. Data are expressed as 2−∆∆Ct. (H–J) p21 and p53 protein levels were analyzed by western blotting. Quantification was performed by densitometry and normalized to β-actin. (K–P) Decorin (DCN), metalloproteinases 1 and 9 (MMP1, MMP9), elastin (ELN), and collagen type 7 alpha 1 (COL7A1) mRNA levels were measured by real-time PCR. Data are expressed as 2−∆∆Ct. All data are expressed as a scatter plot with mean ± standard error of the mean (SEM) of at least two independent experiments (n = 3). Multiple-comparison analysis of variance (ANOVA) was followed by the post hoc Bonferroni test. * p < 0.05 vs. control. SSR: Solar-simulated radiation.

Article Snippet: Then, membranes were incubated with 5% bovine serum albumin (BSA) for 2 h and labeled overnight at 4 ◦C with the antibodies p21 (NB100-1941, Novus Biologicals, Centennial, CO, USA) and p53 (18032, Cell Signaling, Danvers, MA, USA).

Techniques: Irradiation, LDH Cytotoxicity Assay, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Comparison, Control

Journal: International journal of molecular sciences

Article Title: Obacunone Photoprotective Effects against Solar-Simulated Radiation-Induced Molecular Modifications in Primary Keratinocytes and Full-Thickness Human Skin.

doi: 10.3390/ijms241411484

Figure Lengend Snippet: Figure 6. Obacunone prevents SSR-induced p53 and p21 gene and protein expression modulations in a FT skin model. FT skin models were pretreated for 24 h with obacunone (12.5 µM, 25 µM, and 50 µM) and subjected to 18 J/cm2 of SSR. (A) 24 h after irradiation, p21 and p53 mRNA levels were measured by real-time PCR. Data are expressed as 2−∆∆Ct. (B) p21 and p53 levels were analyzed by Western blotting. Quantification was performed by densitometry and normalized to β-actin. Data are expressed as 2−∆∆Ct. All data are expressed as a scatter plot with mean ± standard error of the mean (SEM) of at least two independent experiments (n = 3). Multiple-comparison analysis of variance (ANOVA) was followed by the post hoc Bonferroni test. * p < 0.05 vs. control. # p < 0.05 vs. SSR stimulus. OBA: obacunone.

Article Snippet: Then, membranes were incubated with 5% bovine serum albumin (BSA) for 2 h and labeled overnight at 4 ◦C with the antibodies p21 (NB100-1941, Novus Biologicals, Centennial, CO, USA) and p53 (18032, Cell Signaling, Danvers, MA, USA).

Techniques: Expressing, Irradiation, Real-time Polymerase Chain Reaction, Western Blot, Comparison, Control