Journal: Aging Cell

Article Title: Transcriptional Profiling at Single‐Cell Resolution Reveals Diversity and Regulatory Networks of Primary and Secondary Senescent Cells

doi: 10.1111/acel.70540

Figure Lengend Snippet: Transcriptional heterogeneity and lineage‐resolved progression in primary senescence at single‐cell level. (A) Experimental overview. Renal epithelial cells were irradiated (IR; 10 Gy, 10 days) to induce primary senescence, with quiescent controls (QUI; 0.01% serum, 3 days) processed for scRNA‐seq. (B) Expression levels of senescence and SASP‐related genes in senescent relative to the controls (QUI, n = 3; IR, n = 3). (C) Secreted IL‐6 levels in CM measured using ELISA (QUI, n = 6; IR, n = 6). Data are presented as the means ± the standard error of the mean (unpaired two‐tailed t ‐test; * p < 0.05, ** p < 0.01, *** p < 0.001). (D) UMAP of primary dataset showing clusters grouped into non‐senescent (C4 and C9), intermediate (C0, C1, C3, and C7), and fully senescent states (C5, C6, and C8) (left). Each bar represents either IR or QUI, and each colored segment's height indicates the fraction of one of the three senescence states within that group (middle). Stacked bar chart showing the proportions of IR and QUI cells across each cluster (right). (E) Feature plots showing expression levels of proliferation and senescence‐associated genes. (F) Heatmap of pathway activity across clusters scored via gene set variation analysis, with Z ‐score normalization. (G) UMAP trajectory analysis using Slingshot identifying three senescence progression lineages. Trajectory lines overlaid on UMAP. Cell clusters are colored by pseudotime progression. (H, I) Boxplots of normalized pathway scores for DNA repair (H) and SASP‐related gene sets (I) across clusters (Kruskal–Wallis test, with pairwise Wilcoxon rank‐sum test; adjusted p‐values as shown). (J) Enriched pathways of non‐senescent, intermediate, and fully senescent states in the primary SnCs. p‐values were calculated using a hypergeometric distribution. (K) TradeSeq‐based heatmap of temporally regulated top 500 genes along the pseudotime trajectory for lineage 3 ( p < 0.05), with representative late‐pseudotime genes highlighted.

Article Snippet: Human renal epithelial cells (ATCC; PCS‐400‐011) were cultured in Renal Epithelial Cell Basal Medium (ATCC; PCS‐400‐030) supplemented with the Renal Epithelial Cell Growth Kit (ATCC; PCS‐400‐040), which maintains the cultures at a final serum concentration of 0.5% and incubated at 37°C in 10% CO 2 and 3% O 2 .

Techniques: Single Cell, Irradiation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Activity Assay

Journal: Aging Cell

Article Title: Transcriptional Profiling at Single‐Cell Resolution Reveals Diversity and Regulatory Networks of Primary and Secondary Senescent Cells

doi: 10.1111/acel.70540

Figure Lengend Snippet: SASP‐driven secondary senescence shows distinct transcriptional states. (A) Experimental overview: Proliferative renal epithelial cells were treated with CM from quiescent cells (QCMT) or primary senescent cells (SCMT) and separately processed for scRNA‐seq. (B) qPCR validation of senescence/SASP‐associated genes and expressed as fold changes in SCMT versus QCMT (QCMT, n = 4; SCMT, n = 3). Data are presented as the mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐tailed unpaired t ‐test) (C) Secreted IL‐6 levels in CM measured by ELISA (QCMT, n = 12; SCMT, n = 8). (D) UMAP of secondary SnCs showing clusters grouped into non‐senescent (C2 and C6), intermediate (C0, C1, and C4), and fully senescent clusters (C3, C5, and C7) (left). Each bar represents either QCMT or SCMT, and each colored segment's height indicates the fraction of one of the three senescence states within that group (middle). Stacked bar chart showing the proportions of QCMT and SCMT cells across each cluster (right). (E) Feature plots of representative proliferation and senescence‐associated genes across clusters. (F) Heatmap of pathway activities across clusters ( Z ‐score normalized). (G) UMAP trajectory analysis using Slingshot identifies four lineages with distinct terminal clusters, including a senescence‐resistant endpoint. Trajectory lines indicate senescence progression, and clusters are colored by pseudotime. (H, I) Boxplots of DNA repair (H) and SASP‐related gene set scores (I) across clusters (Kruskal–Wallis two‐sided test with pairwise Wilcoxon rank‐sum test; adjusted p‐values as shown). (J) Enriched pathways categorized into non‐senescent, intermediate, and fully senescent states. p‐values were calculated using a hypergeometric distribution. (K) Heatmap displaying temporally regulated the top 500 genes identified through tradeSeq along the pseudotime trajectory for lineage 4 in secondary senescence (hypergeometric distribution; p < 0.05).

Article Snippet: Human renal epithelial cells (ATCC; PCS‐400‐011) were cultured in Renal Epithelial Cell Basal Medium (ATCC; PCS‐400‐030) supplemented with the Renal Epithelial Cell Growth Kit (ATCC; PCS‐400‐040), which maintains the cultures at a final serum concentration of 0.5% and incubated at 37°C in 10% CO 2 and 3% O 2 .

Techniques: Biomarker Discovery, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Elucidating the therapeutic efficacy and mechanisms of arctigenin in ameliorating renal fibrosis: a combined transcriptomic and proteomic study

doi: 10.3389/fphar.2026.1796732

Figure Lengend Snippet: ATG ameliorates UUO-induced RF in rats by modulating the S100A8/A9/NOX/NF-κB signaling pathway, and additionally attenuates TGF-β1-induced fibrotic responses in HK-2 cells. (A–F) Relative mRNA expression levels of key genes (S100A8, S100A9, NOX2, NOX4, IκBα, and NF-κB p65) in the S100A8/A9/NOX/NF-κB pathway were measured via RT-qPCR in renal tissues. (G–L) Protein expression levels of S100A8, S100A9, NF-κB p65, and phosphorylated NF-κB p65 were detected using Western blot, including representative bands and quantitative analysis. (M) Cell viability of HK-2 cells under varying concentrations of ATG was assessed via CCK-8 assay. (N) Cell viability under different concentrations of PAQ was similarly evaluated using CCK-8. (O) Changes in HK-2 cell viability before and after drug treatment were determined via CCK-8. (P-R) mRNA expression levels of α-SMA, collagen I, and fibronectin in cells were quantified through RT-qPCR. (S–U) Western blot results and quantitative analysis of α-SMA and vimentin protein expression in cells. (V–W) Representative images of wound healing assays at 0 h and 24 h (scale bar = 100 μm) along with quantitative analysis of cell migration rates. Data are presented as mean ± SEM, n = 3 per group ( n = 6 per group for A-F), * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant.

Article Snippet: Human renal cortical proximal tubular epithelial HK-2 cells (STCC10303P, Servicebio) were maintained in a humidified incubator (Thermo Fisher; MA, USA) at 37 °C with 5% CO 2 , and cultured in DMEM/F-12 medium (G4612) supplemented with 10% fetal bovine serum (G8003) and 1% penicillin-streptomycin (G4003).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Migration

Journal: Frontiers in Pharmacology

Article Title: Elucidating the therapeutic efficacy and mechanisms of arctigenin in ameliorating renal fibrosis: a combined transcriptomic and proteomic study

doi: 10.3389/fphar.2026.1796732

Figure Lengend Snippet: ATG alleviates TGF-β1-induced fibrosis in HK-2 cells by regulating the S100A8/A9/NOX/NF-κB signaling pathway. (A–F) Relative mRNA expression levels of key genes (S100A8, S100A9, NOX2, NOX4, IκBα, and NF-κB p65) in the S100A8/A9/NOX/NF-κB signaling pathway were measured in cells using RT-qPCR. (G–L) Relative protein expression levels of key proteins (S100A8, S100A9, NF-κB p65, and phosphorylated NF-κB p65) in the S100A8/A9/NOX/NF-κB signaling pathway were detected by Western blot, including representative band images and quantitative analysis. (M,N) Immunofluorescence images of NOX2 in cells and quantitative analysis of relative fluorescence intensity. (O,P) Immunofluorescence images of IKKβ in cells and quantitative analysis of relative fluorescence intensity. (Q,R) Immunofluorescence images of IκBα in cells and quantitative analysis of relative fluorescence intensity (scale bar = 50 μm). Data are presented as mean ± SEM, n = 3 per group, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant.

Article Snippet: Human renal cortical proximal tubular epithelial HK-2 cells (STCC10303P, Servicebio) were maintained in a humidified incubator (Thermo Fisher; MA, USA) at 37 °C with 5% CO 2 , and cultured in DMEM/F-12 medium (G4612) supplemented with 10% fetal bovine serum (G8003) and 1% penicillin-streptomycin (G4003).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Elucidating the therapeutic efficacy and mechanisms of arctigenin in ameliorating renal fibrosis: a combined transcriptomic and proteomic study

doi: 10.3389/fphar.2026.1796732

Figure Lengend Snippet: ATG ameliorates UUO-induced RF and TGF-β1-induced fibrosis in HK-2 cells by regulating the S100A8/A9/NOX/NF-κB signaling pathway, which modulates TCA cycle and oxidative phosphorylation disruptions, thereby suppressing inflammation and oxidative stress-driven EMT. (A–C) Expression levels of TCA cycle-related factors (CA, NAD-MDH, NAD-ME) in renal tissues. (D–F) Expression levels of oxidative phosphorylation-related factors (ATP, CK, NADK) in renal tissues. (G–I) Relative mRNA expression levels of inflammatory factors (TNF-α, IL-6, IL-1β) in renal tissues detected by RT-qPCR. (J–L) Expression levels of oxidative stress markers (MDA, SOD, GSH) in renal tissues. (M–O) Relative mRNA expression levels of EMT markers (E-cadherin, N-cadherin, Vimentin) in renal tissues detected by RT-qPCR. (P–R) Expression levels of TCA cycle-related factors (CA, NAD-MDH, NAD-ME) in HK-2 cells. (S–U) Expression levels of oxidative phosphorylation-related factors (ATP, CK, NADK) in HK-2 cells. (V–X) Relative mRNA expression levels of inflammatory factors (TNF-α, IL-6, IL-1β) in HK-2 cells detected by RT-qPCR. (Y-AA) Relative levels of ROS in HK-2 cells measured by flow cytometry. (AB-AD) Relative mRNA expression levels of EMT markers (E-cadherin, N-cadherin, Vimentin) in HK-2 cells detected by RT-qPCR. Data are presented as mean ± SEM, n = 6 per group ( n = 3 per group for P-AD), * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant.

Article Snippet: Human renal cortical proximal tubular epithelial HK-2 cells (STCC10303P, Servicebio) were maintained in a humidified incubator (Thermo Fisher; MA, USA) at 37 °C with 5% CO 2 , and cultured in DMEM/F-12 medium (G4612) supplemented with 10% fetal bovine serum (G8003) and 1% penicillin-streptomycin (G4003).

Techniques: Phospho-proteomics, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: bioRxiv

Article Title: Development of high-affinity, single-domain protein binders for neutralizing household allergens

doi: 10.1101/2025.08.03.668213

Figure Lengend Snippet: (a) Thermostability profile of AVA-1-1-C3 as a representative protein, as determined by differential scanning fluorimetry. (b-g) Cytotoxicity of primary human cells following 48-hour exposure to AVA-1-1-C3 or an equivalent concentration of bovine serum albumin (BSA), evaluated using the CellTiter-Glo® (CTG) luminescent cell viability assay. Cell types included human epithelial keratinocytes (HEK, b), human dermal fibroblasts (HDF, c), human follicle dermal papilla cells (HFDPC, d), human renal cortical epithelial cells (HRCEPC, e), human skeletal muscle cells (HSKMC, f), and human dermal microvascular endothelial cells (HDMEC, g). No significant differences in cell viability were observed. Data in (b-g) represents mean ± s.d. and are representative of two experimental replicates.

Article Snippet: For in vitro toxicity studies, primary cells including human dermal fibroblasts (HDF, Cell Applications, Cat# 106K-05a, lot 1632), human epidermal keratinocytes (HEK, Cell Applications, Cat# 102-05a, lot 2146), human dermal microvascular endothelial cells (HDMEC, PromoCell, Cat# C-12210, lot 483Z001.3), human skeletal muscle cells (HSKMC, Cell Applications, Cat# 150K-05a, lot 3507), and human renal cortical epithelial cells (HRCEpC, Promocell, Cat# C-12660, lot 501Z019.23).

Techniques: Concentration Assay, Cell Viability Assay