Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates osteoarthritis progression through p53-dependent autophagy activation: evidence from network analysis and experimental validation

doi: 10.3389/fphar.2026.1686555

Figure Lengend Snippet: PT modulates p53 localization, AMPK/mTOR signaling, and autophagy-related markers in chondrocytes. (A) CETSA results assessing the thermal stability of p53 in C28/I2 cells treated with PT or vehicle across a temperature range of 38 °C–70 °C. Representative WB bands are shown above the normalized thermal aggregation curves; (B) Representative immunofluorescence confocal microscopy images illustrating p53 subcellular localization (red) and DAPI-stained nuclei (blue) in chondrocytes across different treatment groups (scale bar = 10 μm); (C) Representative WB bands showing p53 distribution (nuclear, cytoplasmic, and total), the phosphorylation status of AMPK and mTOR, and autophagy markers (LC3 and p62); (D–K) Quantitative analyses of protein expression levels: total p53/GAPDH (D) , nuclear p53/PCNA (E) , cytoplasmic p53/GAPDH (F) , nuclear/cytoplasmic p53 ratio (G) , p-AMPK/AMPK (H) , p-mTOR/mTOR (I) , p62/GAPDH (J) , and the LC3II/I ratio (K) ; (L) Representative TUNEL staining images showing apoptotic cells (red) and nuclei (blue) (scale bar = 50 μm); (M) Quantitative analysis of the percentage of TUNEL-positive cells across groups. Data = mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 vs. control; ###P < 0.001 vs. IL-1β; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. IL-1β + PT.

Article Snippet: C28/I2 cells were treated with PT (HY-N0828, Lot# 130513, MedChemExpress, Shanghai, China) without further purification.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Phospho-proteomics, Expressing, TUNEL Assay, Control

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: In contrast to SAL, C28 reduces AR nuclear translocation associated with inhibition of phosphorylation of both AR and HSP27. A: Immunofluorescence using anti-AR antibody to analyze the intracellular localization of AR (red) in C4-2 cells treated with SAL (1 nM R1881) , C28 (30 µM), or DMSO as solvent control. Samples without the primary antibody serve as a negative control. DAPI staining was used to detect nuclei (blue), anti-wheat germ agglutinin antibody is used to detect cell membrane (WGA, green). Scale bars represent 10 µm. B: Quantification of the integrated density of AR in the nuclei. Data are presented as mean ± SEM (n = 10 technical replicates). C: Western blot analysis of treated C4-2 cells with AR-antagonists with increasing concentrations of C28 (3, 10, and 30 µM) and SAL analyzing levels of AR, pAR (Ser81), HSP27, and pHSP27 with β-Actin that serves as loading control (N = 3). The numbers indicate the band intensities normalized to β-Actin and relative to DMSO control. D and E: mRNA levels of AR-target genes NKX3.1 and FKBP5 measured by qRT-PCR. D: C4-2 cells, bar graphs are shown as mean ± SEM from nine technical replicates (n = 9) of three independent experiments. E: PC3 cells, bar graphs are shown as mean ± SEM from six technical replicates (n = 6) of two independent experiments. TBP and alpha-Tubulin were used as housekeeping genes for normalization. F: Immunoprecipitation of AR and detection of AR. G: Co-Immunoprecipitation (Co-IP) of AR and detection of HSP27. Cytosol (C), Nucleus (N). (N = 3). Band intensities are shown relative to the cytosol. Statistical analysis was performed using a two-tailed unpaired Student t -test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques: Translocation Assay, Inhibition, Phospho-proteomics, Immunofluorescence, Solvent, Control, Negative Control, Staining, Membrane, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: C28 induces cellular senescence and inhibits growth of CRPC adherent culture, 3D tumor spheroids and xenografted CRPC tumors in mice. A: 9 days growth curve of adherent C4-2 cells treated with AR-ligands. Crystal violet absorbance at OD 590 nm was normalized to day 0. Bar graphs are shown as mean ± SEM from four technical replicates (n = 4) of two independent experiments. B: Percentage of SA β-Gal positive C4–2 cells. Data are presented as mean ± SEM from twelve biological replicates (n = 12) of three independent experiments. C: Generation and treatment of C4-2 tumor spheroids with C28 (30 µM) or DMSO as a solvent control for 15 days. Scale bars indicate 10 µm. D: Reduction of tumor spheroid volume by C28 treatment. Bar graphs are shown as mean ± SEM from twenty technical replicates (n = 20) of two independent experiments. E: Ki67 immunofluorescence staining, as a proliferation marker, and SA β-Gal activity staining as a cellular senescence marker in C4-2 spheroids. Samples without primary antibody serve as a negative control. Scale bars indicate 10 µm. F: Quantification of Ki67 positive signals per area. Bar graphs are shown as mean ± SEM from ten technical replicates (n = 10) of two independent experiments. G: qRT-PCR analysis of CDKN2B and CDKN2A , encoding p15 INK4b and p16 INK4a , respectively in C4-2 spheroids from six technical replicates (n = 6) of two independent experiments. H: Growth responses of C4-2 xenografts in mice to a 20-day course of C28 (100 mg/kg/day, N = 6), DHT (SAL, 50 mg/kg/day, N = 7), or vehicle control (0.5 % Tween 80, N = 5). Tumor volume was measured during treatment, showing growth inhibition by C28 and DHT (SAL). I: Mice body weight was measured every two days during the treatment period. J: Ki67 and SA β-Gal staining of C4-2 xenograft tumor slices from C28, DHT (SAL), or vehicle-treated mice. Scale bars indicate 10 µm. K: Quantification of Ki67 positive signals per area in CRPC xenograft tumors. L: qRT-PCR analysis of CDKN2B and CDKN2A in xenograft tumors treated with C28 (N = 6) or DHT (N = 7) compared to vehicle control (N = 5). TBP and alpha-Tubulin were used as housekeeping genes for normalization in all qRT-PCR analysis. Two-way ANOVA was used for analyses of growth curves and volume of the tumors, and two-tailed unpaired Student t -test was used for comparing treatments with the control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques: Solvent, Control, Immunofluorescence, Staining, Marker, Activity Assay, Negative Control, Quantitative RT-PCR, Inhibition, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: Both C28 and Sal upregulate p130 levels. A: Western blot analysis of pRb, phospho-pRb (ppRb) and p130 in treated C4-2 cells (N = 3). The expression was normalized to β-Actin as loading control. Numbers indicate normalized band intensities relative to DMSO control. B: qRT-PCR examination of RBL2 expression, encoding p130, in CRPC adherent cultures treated with AR-ligands. Bar graphs are shown as mean ± SEM from nine technical replicates (n = 9) of three independent experiments. C : qRT-PCR analysis of RBL2 expression in CRPC 3D spheroids treated with C28 or DMSO from six technical replicates (n = 6) of two independent experiments. D: qRT-PCR of RBL2 expression in xenograft tumors of mice treated with C28 (N = 6), DHT (SAL, N = 7), or vehicle control (N = 5). TBP and alpha-Tubulin were used as housekeeping genes for normalization. E: Protein levels of p130 in xenograft tumors were analyzed by Western blotting. Numbers show band intensities normalized to β-Actin relative to the vehicle control. F: Protein levels are represented as mean ± SEM values. G: ChIP-seq data indicates AR occupancy at RBL2 in C4-2 cells. H: ChIP-seq data indicates p130 and E2F1 occupancy at the regulatory regions of RBL2 in C4-2 cells. IGV software was used for visualization. I: AR-p130 Co-IP immunoblot of lysates from AR and IgG immunoprecipitations using protein extracts from C4-2 cells treated with C28, SAL or DMSO for 72 h. A two-tailed unpaired Student t -test was used for comparing each treatment with the control.

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, ChIP-sequencing, Software, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: Both AR-agonist and −antagonists downregulate DREAM complex target genes. A: qRT-PCR analysis of the DREAM protein complex regulated genes ATAD2 , AURKB , CCNA2 , MYBL2 and FOXM1 in C4-2 cells treated with C28 (30 µM), Dar (10 µM), SAL or DMSO for 72 h. Bar graphs are shown as mean ± SEM from nine technical replicates (n = 9) of three independent experiments. B : RNA-seq gene expression analysis of DREAM complex gene targets in C4-2 cells treated with SAL. C: qRT-PCR analysis of the selected genes expression in C4-2 spheroids from six technical replicates (n = 6) of two independent experiments. D: qRT-PCR determination of DREAM complex target mRNA levels in C4-2 xenograft tumors treated with C28 (N = 6), DHT (SAL, N = 7), or vehicle control (N = 5). TBP and alpha-Tubulin were used as housekeeping genes for normalization. E: RNA-seq gene expression analysis of DREAM complex targets in C4-2 xenograft tumors treated with vehicle or SAL. All analyzed genes are significantly downregulated ( p < 0.05 ), except ATAD2 which shows a downward trend with p = 0.06. F: AURKB immunofluorescence staining of C4-2 xenograft tumor slices from C28, DHT (SAL), or vehicle-treated mice, with N = 5 for each treatment group. Scale bars indicate 10 µm. G: Quantification of AURKB positive signals per area in CRPC xenograft tumors. A two-tailed unpaired Student t -test was used for comparing each treatment with the control.

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques: Quantitative RT-PCR, RNA Sequencing, Gene Expression, Expressing, Control, Immunofluorescence, Staining, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: DYRK1A inhibition counteracts both cellular senescence induced by C28 or SAL and C28-mediated repression of DREAM target genes. A: Percentage of SA β-Gal positive cells in C4-2 treated cells with AZ191 (800 nM), a DYRK1A inhibitor (DYRK1A-i; AZ191), either alone or in combination with AR-ligands. Bar graphs are shown as mean ± SEM from twelve technical replicates (n = 12) of three independent experiments. B: Growth assay analysis over 9 days in C4-2 treated cells with or without DYRK1A-i (AZ) using crystal violet staining from four technical replicates (n = 4) of two independent experiments. C: Percentage of SA β-Gal positive cells in C4-2 cells transfected with siDYRK1A following AR-ligand treatments from twelve technical replicates (n = 12) of three independent experiments. D: Efficiency of DYRK1A knockdown (siDYRK1A). E: qRT-PCR to analyze the expression of DREAM complex target genes ( ATAD2 , AURKB , CCNA2 , MYBL2 and FOXM1 ) in C4-2 cells treated with the DYRK1A inhibitor AZ191 (DYRK1A-i, 800 nM) either alone or in combination with C28, Dar, SAL or DMSO, relative to DMSO. Bar graphs are shown as mean ± SEM from six technical replicates (n = 6) of two independent experiments. F: qRT-PCR analysis of DREAM complex target gene expression in C4-2 cells transfected with si-mediated knockdown of DYRK1A (siDYRK1A) following treating with C28, Dar, SAL or DMSO, relative to siControl DMSO from nine technical replicates (n = 9) of three independent experiments. TBP and alpha-Tubulin were used as housekeeping genes for normalization. Two-way ANOVA was used for all analysis for growth curves analyses, and a two-tailed unpaired Student t -test was used to compare each treatment with the control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques: Inhibition, Growth Assay, Staining, Transfection, Knockdown, Quantitative RT-PCR, Expressing, Targeted Gene Expression, Two Tailed Test, Control

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: CCNG2 is co-expressed with AR and RBL2 and mediates cellular senescence induced by C28 or SAL . A: Correlation analysis between RBL2 and CCNG2 gene expression in PCa using GEPIA dataset. Values represent spearmen correlation coefficient, p < 0.05. B: Correlation analysis between AR and CCNG2 gene expression in patients with PCa using GEPIA dataset. Values represent spearmen correlation coefficient. p < 0.05. C: qRT-PCR examination of CCNG2 mRNA levels in treated C4-2 cells with C28, Dar, SAL or DMSO. Bar graphs are shown as mean ± SEM from nine technical replicates (n = 9) of three independent experiments. D: qRT-PCR analysis of CCNG2 mRNA levels in C28 treated C4-2 spheroids from six technical replicates (n = 6) of two independent experiments. E: qRT-PCR analysis of CCNG2 mRNA levels in C4-2 xenograft tumors of mice treated with C28 (N = 6), DHT (SAL, N = 7), or vehicle control (N = 5). F: Percentage of SA β-Gal positive cells in transfected C4-2 cells with siCCNG2 following AR-ligand treatments from eight technical replicates (n = 8) of two independent experiments. G: Efficiency of CCNG2 knockdown. Bar graphs are shown as mean ± SEM from six technical replicates (n = 6) of three independent experiments. H: qRT-PCR analysis of CDKN2B mRNA levels in transfected C4-2 cells with siCCNG2 following AR-ligand treatments relative to siControl DMSO from six technical replicates (n = 6) of two independent experiments. For all qRT-PCR analyses, TBP and alpha-Tubulin were used as housekeeping genes for normalization. A two-tailed unpaired Student t -test was performed for statistical analysis.

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques: Gene Expression, Quantitative RT-PCR, Control, Transfection, Knockdown, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: C28 interacts with valine, glutamine and arginine residues of AR being distinct from Dar. A: 2D structure of C28. B: Docking of C28 with AR, calculated affinity: −5.552 kcal/mol. C: 2D structure of Dar. D: Docking of Dar with AR, calculated affinity: −5.949 kcal/mol.

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques:

Journal: Journal of Advanced Research

Article Title: Induction of cellular senescence by androgen receptor agonist or antagonist is mediated via two novel common DYRK1A-DREAM and cyclin G2 signaling pathways in castration-resistant prostate cancer

doi: 10.1016/j.jare.2025.05.019

Figure Lengend Snippet: C28 induces cellular senescence via two pathways. On one hand by AR-p130-DYRK1A-DREAM complex and the other hand by upregulation of CCNG2 signaling. C28- or androgen-bound AR, at SAL, interacts with p130 leading to stabilization of p130 and promoting the formation of the p130-AR protein complex. DYRK1A activation leads to DREAM complex assembly to induce cellular senescence. In common, C28 and SAL also upregulate CCNG2 expression further contributing to the induction of cellular senescence. The figure was in part generated using Biorender.

Article Snippet: Toxicity analysis using PROTOX-3.0 classifies C28 to have moderate to mild toxicity with potential neurotoxicity due to its ability to cross the blood–brain barrier.

Techniques: Activation Assay, Expressing, Generated