Journal: Annals of the rheumatic diseases

Article Title: RUNX1 is expressed in a subpopulation of dermal fibroblasts and is associated with disease severity of systemic sclerosis

doi: 10.1016/j.ard.2025.10.033

Figure Lengend Snippet: TGF- β 1 increases the RUNX1 expression in SSc fibroblasts and inhibition of RUNX1 reduces ECM markers. (A) Western blot of 3 isolated fibroblasts lines treated with TGF- β 1. The blot shows all isoforms of RUNX1a, b, and c that are overexpressed under the TGF- β 1 stimulation. (B) Schematic graph illustrating the timeline for the culture and TGF- β 1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and normal human dermal (NHD) fibroblast cells. RUNX1 expression rate in samples treated with TGF- β 1 (in red) vs control (in blue) for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the 2 SSc-isolated fibroblast lines at 12 hours after exposure vs the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF- β 1 treatment vs the baseline. Data from B to D were obtained through publicly available data of GSE12493 . (E) Schematic graph showing 2 lines of SSc-isolated fibroblasts treated with siRNA against RUNX1 (siRUNX1) and nontargeting control siRNA (siNC). (F) UMAP projection and dot plot of RUNX1 and CBFB of the single-cell RNA-seq data. (G) UMAP of 10 fibroblast clusters (0–9) for siR-UNX1 and siNC. (H) Cell proportion of siRUNX1 and siNC per cluster. (I) Top 4 upregulated and downregulated marker genes per cluster. (J) Top 15 enriched pathways that are significantly represented across siRUNX1 and siNC (K) Bar plots showing the percentage of cells expressing COL1A1, FN1, COL4A1, LUM, ACTA2, LGR5, COL8A1, COMP , and THBS1 per condition (red: siRUNX1, green: siNC) or per cluster. (L) Module score for extracellular matrix organisation pathway per cluster and per condition. (M) Feature plot of the ECM module score. dcSSc, diffuse cutaneous SSc; DEG, differentially expressed gene; ECM, extracellular matrix; RUNX1, runt-related transcription factor 1; siRUNX1, siRNA targeting RUNX1; SSc, systemic sclerosis; TGF- β , transforming growth factor- β ; UMAP, uniform manifold approximation and projection.

Article Snippet: We then analysed a previously generated DNA microarray dataset (National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO): GSE12493 ) consisting of 2 independent SSc fibroblast cell lines, 1 healthy control fibroblast cell line (isolated in parallel), and 1 normal human dermal (NHD) fibroblast cell line obtained from American Type Culture Collection (ATCC), treated with 50 pM TGF- β 1 [ ] ( ).

Techniques: Expressing, Inhibition, Western Blot, Isolation, Control, Single Cell, RNA Sequencing, Marker

Journal: Annals of the rheumatic diseases

Article Title: RUNX1 is expressed in a subpopulation of dermal fibroblasts and is associated with disease severity of systemic sclerosis

doi: 10.1016/j.ard.2025.10.033

Figure Lengend Snippet: RUNX1 contributes to fibroblast activation, proliferation and contraction. (A) RUNX1 western blot of CRISPR-generated RUNX1 KO and wild-type (WT) fibroblasts under the TGF- β 1 stimulation vs control. RUNX1 isoforms of a, b, and c were marked in the blot by arrows. (B) α -SMA and RUNX1 IF staining of KO and WT fibroblasts under the TGF- β 1 stimulation vs control. (C) α -SMA western blot of KO and WT fibroblasts under the TGF- β 1 stimulation vs control. (D) ACTA2 mRNA expression of KO and WT fibroblasts under the TGF- β 1 induction vs control. (E) Fold change expression of FN1, COL1A1, LUM , and SFRP4 in TGF- β 1-induced SSc fibroblasts treated with Ro5–3335 compared to control (3 lines of SSc fibroblasts, 2 replicates each). (F) Proliferation curve of normal human dermal (NHD) fibroblasts in the presence and absence of Ro5–3335. (G,H) The 3D collagen contraction assays, fixed (G) and floating (H) models, of NHD fibroblasts treated with Ro5–3335 (4 replicates for each condition). SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. Negative control is collagen matrix with no fibroblasts. The overhead pictures represent 1 replicate for each condition. (I) 3D self-assembled (SA) tissue constructs from the healthy- and SSc-isolated fibroblast lines with donors’ clinical characteristics. H&E staining of representative untreated and Ro5–3335-treated tissues. (J) Tissue area fold change of each cell line over the control for healthy and SSc SA tissues. Data from 3 healthy and 4 SSc lines, 3 replicates per line, repeated in 2 independent sets. (K) Change in area of an SSc-isolated SA tissue when treated for 1, 2, or 3 weeks with Ro5–3335 compared to control (Student’s t test P value: **.001-.01, ****<.0001 in GraphPad Prism v9). α -SMA, alpha smooth muscle actin; H&E, haematoxylin and eosin; KO, knockout; RUNX1, runt-related transcription factor 1; SSc, systemic sclerosis; TGF- β , transforming growth factor- β ;Clustered Regularly Interspaced Palindromic Repeats (CRISPR),Smad Family Member 3 (SMAD3),Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Not Applicable (N/A), Quantitative Polymerase Chain Reaction (QPCR), Quantitative Polymerase Chain Reaction, Immunofluorescenc (IF).

Article Snippet: We then analysed a previously generated DNA microarray dataset (National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO): GSE12493 ) consisting of 2 independent SSc fibroblast cell lines, 1 healthy control fibroblast cell line (isolated in parallel), and 1 normal human dermal (NHD) fibroblast cell line obtained from American Type Culture Collection (ATCC), treated with 50 pM TGF- β 1 [ ] ( ).

Techniques: Activation Assay, Western Blot, CRISPR, Generated, Control, Staining, Expressing, Positive Control, Negative Control, Construct, Isolation, Knock-Out, Real-time Polymerase Chain Reaction

Journal: Pharmaceuticals

Article Title: Ethanolic Fenugreek Extract: Its Molecular Mechanisms against Skin Aging and the Enhanced Functions by Nanoencapsulation

doi: 10.3390/ph15020254

Figure Lengend Snippet: Anti-aging activities’ evaluation of fenugreek extract: ( a ) In vitro collagenase inhibition of fenugreek extract (denoted as Extract) and liponiosome encapsulating fenugreek extract (denoted as LNF). ( b ) Effect of fenugreek extract on collagen production. Human dermal fibroblast cells were treated with 125 µg/mL of fenugreek extract and rutin, and 50 µg/mL of vitamin C as a positive control and 0.005% DMSO as a control (vehicle), for 7 and 14 days. Data are reported as means ± SD ( n = 3). *, p < 0.05; ***, p < 0.001.

Article Snippet: Human dermal fibroblast cells were purchased from the American Type Culture Collection: ATCC (PCS-201-010) and immortalized human keratinocytes (HaCaT) were purchased from Cell Lines Service, Germany (Cat. No. 300493).

Techniques: In Vitro, Inhibition, Positive Control, Control

Journal: Pharmaceuticals

Article Title: Ethanolic Fenugreek Extract: Its Molecular Mechanisms against Skin Aging and the Enhanced Functions by Nanoencapsulation

doi: 10.3390/ph15020254

Figure Lengend Snippet: Evaluation of LNF activities: ( a ) Cytotoxicity induced by LNF: The effect of blank, LNF, and fenugreek extract on cell viability. Human dermal fibroblast cells were treated with different concentrations of blank (liponiosome without fenugreek extract denoted as Blank), LNF, and fenugreek extract for 24 h. Data are means ± SD ( n = 3). ( b ) Collagen production induced by LNF: The effect of LNF on collagen production. Human dermal fibroblast cells were treated with 0.005% DMSO as a control (vehicle), 7 µg/mL of extract, and 100 µg/mL of LNF (7 µg/mL of extract equivalence), together with blank particles, for 7 and 14 days. Data are represented as means ± SD ( n = 3). *, p < 0.05.

Article Snippet: Human dermal fibroblast cells were purchased from the American Type Culture Collection: ATCC (PCS-201-010) and immortalized human keratinocytes (HaCaT) were purchased from Cell Lines Service, Germany (Cat. No. 300493).

Techniques: Control

Journal: Pharmaceuticals

Article Title: Ethanolic Fenugreek Extract: Its Molecular Mechanisms against Skin Aging and the Enhanced Functions by Nanoencapsulation

doi: 10.3390/ph15020254

Figure Lengend Snippet: Inhibition of UV-induced MMPs and interleukin secretion on co-cultured skin cells by fenugreek extract and LNF: ( a ) Effect of UV-induced cytotoxicity after the pretreatments of resveratrol, rutin, fenugreek extract, blank nanoparticles (liponiosome without fenugreek extract), LNF nanoparticles, and 0.005% DMSO as a control (vehicle). HaCAT and human dermal fibroblast cells were co-cultured and pretreated with 7 µg/mL of extract and 100 µg/mL of LNF (7 µg/mL of extract equivalence), together with 100 µg/mL of blank, 10 µg/mL of resveratrol, 7 µg/mL of rutin as a positive, and 0.005% of DMSO as a control (vehicle), for 24 h before UV exposure. Data are means ± SD ( n = 3). *, p < 0.05. ( b ) The levels of UV-induced MMP1 and MMP9 secretions after fenugreek extract and LNF treatments. Data are reported as means ± SD ( n = 3). *, p < 0.05. ( c ) The levels of UV-induced IL-6 and IL-8 secretions after fenugreek extract and LNF treatments. Data are reported as means ± SD ( n = 3). *, p < 0.05.

Article Snippet: Human dermal fibroblast cells were purchased from the American Type Culture Collection: ATCC (PCS-201-010) and immortalized human keratinocytes (HaCaT) were purchased from Cell Lines Service, Germany (Cat. No. 300493).

Techniques: Inhibition, Cell Culture, Control

Journal: Molecules

Article Title: Targeting Hydroxybenzoic Acids to Mitochondria as a Strategy to Delay Skin Ageing: An In Vitro Approach

doi: 10.3390/molecules27196183

Figure Lengend Snippet: Effects of hydroxybenzoic-based mitochondriotropic antioxidants MB1-MB4 on cell mass ( A , D ), metabolic activity ( B , E ), and intracellular ATP ( C , F ) of normal human dermal fibroblasts (NHDF). Normal human dermal fibroblasts were treated with increasing concentrations of the different molecules for 48 h. Cellular mass, metabolic activity, and ATP levels were evaluated using sulforhodamine B (SRB) assay, resazurin reduction assay, and CellTiter-Glo Luminescent Cell Viability Assay, respectively. Data are the mean ± SE of four independent experiments. The results are expressed as a percentage of the control. Statistically significant differences between control (CTL) and treated groups were evaluated using a t -test. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 compared to the respective control (CTL, vehicle-treated cells), with the colours green, orange, blue and pink to correspond to each compound MB1, MB2, MB3 and MB4, respectively.

Article Snippet: Normal human dermal fibroblasts (NHDF), with batch n° 1839 and reference 106-05a, were purchased from Cell Applications, Inc (San Diego, CA, USA).

Techniques: Activity Assay, Sulforhodamine B Assay, Cell Viability Assay, Control

Journal: Molecules

Article Title: Targeting Hydroxybenzoic Acids to Mitochondria as a Strategy to Delay Skin Ageing: An In Vitro Approach

doi: 10.3390/molecules27196183

Figure Lengend Snippet: Effects of hydroxybenzoic-based mitochondriotropic antioxidants MB1-MB4 on DNA damage. Normal Human Dermal Fibroblasts (NHDF) were treated with MB1, MB2, and MB4 at 25 μM and MB3 at 6.3 μM for a period of 48 h. DNA damage was assessed using the comet assay. H 2 O 2 at 500 μM was used as a positive control. The different parameters of Comet Height ( A ), Comet Length ( B ), Tail DNA % ( C ), Tail Length ( D ) and Tail Moment ( E ) in NHDF were quantified using Cell Profiler software. Results are expressed as a function of control (CTL = 100%) and represent the interquartile range (Q1–Q3), together with the (⦁) mean and (⎯) median of three independent experiments. Data outside the Q1–Q3 range are represented as data outliers (*). Statistically significant differences between control (CTL) and treated groups were evaluated using a t -test. **** p < 0.0001, ** p < 0.01 and * p < 0.05 compared to CTL (vehicle-treated cells).

Article Snippet: Normal human dermal fibroblasts (NHDF), with batch n° 1839 and reference 106-05a, were purchased from Cell Applications, Inc (San Diego, CA, USA).

Techniques: Single Cell Gel Electrophoresis, Positive Control, Software, Control

Journal: Molecules

Article Title: Targeting Hydroxybenzoic Acids to Mitochondria as a Strategy to Delay Skin Ageing: An In Vitro Approach

doi: 10.3390/molecules27196183

Figure Lengend Snippet: Effects of hydroxybenzoic-based mitochondriotropic antioxidants MB1-MB4 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). OCR- and ECAR-associated parameters were assessed with a Seahorse XFe96 Extracellular Flux Analyzer. OCRs ( A ) and ECARs ( B ) were assessed in normal human dermal fibroblast (NHDF) cells treated with MB1, MB2, and MB4 at 25 μM and MB3 at 6.3 μM for 48 h. Several OCR parameters were evaluated: ATP production-linked OCR ( C ), basal respiration ( D ), proton leak-based OCR ( E ), maximal respiration ( F ), non-mitochondrial respiration ( G ) and spare respiratory capacity ( H ). ECAR parameters were also evaluated, including basal ECAR ( I ), stressed ECAR ( J ), and stressed OCR ( K ). Data are the mean ± SE of three independent experiments and the results are expressed in the interquartile range (Q1–Q3) together with the (−) median. Statistically significant differences between control (CTL) and treated groups were evaluated using a t -test. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 compared to the respective CTL (vehicle-treated cells).

Article Snippet: Normal human dermal fibroblasts (NHDF), with batch n° 1839 and reference 106-05a, were purchased from Cell Applications, Inc (San Diego, CA, USA).

Techniques: Control

Journal: AMB Express

Article Title: Repurposing rupatadine as topical treatment against methicillin-resistant Staphylococcus aureus

doi: 10.1186/s13568-025-01947-w

Figure Lengend Snippet: Dose–Response Curves for Cytotoxicity Testing on Human Skin Fibroblasts (HSFs). Dose–response relationships of rupatadine, DMSO (vehicle control), and doxorubicin (positive control) on HSFs cell viability. Cells were treated with increasing concentrations of test compounds for 2 h, and cell viability was assessed using the SRB colorimetric assay. IC 50 values were calculated using GraphPad Prism software. The dotted line indicates IC 50 of rupatadine (1150 µg/mL). Rupatadine demonstrated minimal cytotoxicity compared to the potent positive control doxorubicin, while DMSO showed no significant cytotoxic effects across all tested concentrations

Article Snippet: Rupatadine cytotoxicity was assessed using Sulforhodamine B (SRB) colorimetric assay on human skin fibroblasts (HSFs) (AcceGen Biotech Cat. # ABC-TC3451, USA; Obtained in September 2023).

Techniques: Control, Positive Control, Colorimetric Assay, Software

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults reveal reduced cytotoxicity towards senescent fibroblasts. (A) Graphical illustration of the NK cell mediated target cell cytotoxicity assay. Target cells were first stained with calcein acetomethoxymethyl (AM), a vital fluorescent dye. Calcein AM is a non‐fluorescent compound that pass the intact cell membrane into the cytoplasm. Hydrolysis of calcein AM by intracellular esterases in live cells generates calcein, a hydrophilic, intensely fluorescent molecule which reliably stays in the cytoplasm. The stained target cells were next co‐cultured with NK cells isolated from young or old human or mice. NK cells exert their cytotoxicity towards target cells through the release of perforin and granzyme B. Upon lysis of target cells, the calcein dye is released and the loss of the dye is measured as a shift in fluorescence intensity by flow cytometry. Dead cells will appear to the left of the histogram, while alive cells on the right side. The percentage of dead cells can then simply be calculated and presented. (B) Graphical scheme depicts the experimental groups: Co‐cultures of NK cells from young adults with senescent human dermal fibroblasts in the top row and NK cells from old adults with senescent HDF in the bottom row. (C) Histogram (bi‐exponential scale) showing cytotoxicity of NK cells from young and old adults on different senescent HDF. RS, replicative senescent HDF, DIS, doxorubicin induced senescent HDF, IR, ionizing radiation induced senescence, CA, chronologically aged HDF (~75 years). The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (D) The graph depicts the percentage of senescent HDF death ( y ‐axis) by NK cells isolated from young and old human adult. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 5. Two tailed Student's t ‐test was used to assess the significance between young and old groups for each of senescence model. (E) Illustration of the experimental design showing cytotoxic activity of NK cells derived from bone marrow and spleen of young and old mice against aged murine dermal fibroblasts (MDF). (F) Histogram depicting cytotoxicity of NK cells from young and old mice on old MDF. The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (G) The graph depicts the percentage of senescent MDF death ( y ‐axis) by NK cells isolated from young and old mice. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 3. Each mouse NK cell sample used in the cytotoxicity assay was the pool of NK cells isolated from three different mice. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups.

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Cytotoxicity Assay, Staining, Membrane, Cell Culture, Isolation, Lysis, Fluorescence, Flow Cytometry, Two Tailed Test, Activity Assay, Derivative Assay, Comparison

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN restores impairment of conjugation, degranulation and mitochondrial ATP generation in old Natural killer cells. (A) Synapse formation with conjugation of NK cells with the target senescent fibroblasts and the tubulin network pulling the perforin and granzyme B containing vesicles in the direction of the synapse. (B) Fusion of the NK cell derived secretory granules with the presynaptic membrane of NK cells and concomitant exposure of CD107a at the cell membrane and the release of perforin and granzyme B into the synaptic cleft towards the target cell. (C & D) Percentage of NK cell conjugation with senescent HDF when co‐cultured for (C) 60 and (D) 90 min at an effector to target (E:T) cell ratio of 1:1. Data were represented as mean (percentage of cell conjugation) ± SEM, N = 3. (E) Degranulation of NK cells when co‐cultured with senescent HDF for 7 h at an effector to target (E:T) cell ratio of 10:1. Data were represented as mean (mean fluorescence intensity) ± SEM, N = 6. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C, D and E. (F) Seahorse flux analysis showed quantification of ATP generated by NK cells treated with vehicle from young donors and from NK cells treated with either vehicle or CASIN from old donors. The ATP generation either by glycolysis or by oxidative phosphorylation and total ATP was assessed. Data were represented as mean (ATP level) ± SEM, N = 7. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (G) Mitochondrial structure showing the chemical structure of the mitochondrial fluorescent probe JC‐1 that can form J‐aggregates (red) and J‐monomers (green) indicating high and low mitochondrial membrane potential, respectively. (H) Flow cytometry analysis of J‐aggregates (red) and J‐monomers (green) of young NK cells treated with vehicle, old NK cells treated with either vehicle or CASIN. (I) The graph depicts the percentage of J‐aggregates (Q2 population of figure H) of young NK treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (percentage of cell with J‐aggregate) ± SEM, N = 6. (J) Quantification of the ratio of J‐aggregates to J‐monomers from young and old NK treated with vehicle and old NK cells treated with CASIN. Data were represented as mean (ratio) ± SEM, N = 5. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in I and J.

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Conjugation Assay, Derivative Assay, Membrane, Cell Culture, Fluorescence, Comparison, Generated, Phospho-proteomics, Flow Cytometry

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: Human dermal fibroblast , iXCells Biotechnologies , Cat. #10HU‐013.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay