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

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: Journal of Functional Biomaterials

Article Title: The Impact of the Methacrylation Process on the Usefulness of Chitosan as a Biomaterial Component for 3D Printing

doi: 10.3390/jfb15090251

Figure Lengend Snippet: Microscopic imaging of RFP-HDFCs-Neo cells cultured on biomaterials and as a test model of printed constructs. Imaging was performed 24 h and 7 days after preparation of the constructs.. The images were made with an Olympus IX83 microscope (Olympus, PA, USA) in the bright field (BF) and with the use of red fluorescent lights (TRITC-tetramethylrhodamine). Imaging was performed at both 10× (cell-covered biomaterials) and 4× (3D-printed models) objective magnifications. Control: cells cultured under standard conditions (37 °C and 5% CO 2 ); BCH: cells cultured on biomaterial with added chitosan; and BCM: cells cultured on biomaterial with added methacrylated chitosan.

Article Snippet: The RFP Expressing Human Dermal Fibroblasts-Neonatal cell line (RFP-HDFCs-Neo, ANGIO-PROTEOMIE, cAP-0008-NeoRFP, Boston, MA, USA) was used.

Techniques: Imaging, Cell Culture, Construct, Microscopy, Control

Journal: Journal of Functional Biomaterials

Article Title: The Impact of the Methacrylation Process on the Usefulness of Chitosan as a Biomaterial Component for 3D Printing

doi: 10.3390/jfb15090251

Figure Lengend Snippet: Cytotoxicity of biomaterial extract. LDH assay was performed with the use of L-929 (in accordance with standard PN-EN ISO 10993-5:2009) ( A ) and RFP-HDFCs-Neo ( B ) cells. Negative control: cells cultured under standard conditions (37 °C, 5% CO 2 ); positive control: cells cultured under standard conditions (37 °C, 5% CO 2 ) exposed to 0.1% Triton X-100; BCH: cells+ chitosan-biomaterial extracts; and BCM: cells+ methacrylated chitosan-biomaterial extracts.

Article Snippet: The RFP Expressing Human Dermal Fibroblasts-Neonatal cell line (RFP-HDFCs-Neo, ANGIO-PROTEOMIE, cAP-0008-NeoRFP, Boston, MA, USA) was used.

Techniques: Lactate Dehydrogenase Assay, Negative Control, Cell Culture, Positive Control

Journal: Journal of Functional Biomaterials

Article Title: The Impact of the Methacrylation Process on the Usefulness of Chitosan as a Biomaterial Component for 3D Printing

doi: 10.3390/jfb15090251

Figure Lengend Snippet: Cell proliferation after exposure to biomaterial extract. Alamar Blue assay was performed with the use of L-929 (in accordance with standard PN-EN ISO 10993-5:2009) ( A ) and RFP-HDFCs-Neo ( B ) cells. Negative control: cells cultured under standard conditions (37 °C, 5% CO 2 ); positive control: cells cultured under standard conditions (37 °C, 5% CO 2 ) exposed to 0.1% Triton X-100; BCH: cells+ chitosan-biomaterial extracts; and BCM: cells+ methacrylated chitosan-biomaterial extracts.

Article Snippet: The RFP Expressing Human Dermal Fibroblasts-Neonatal cell line (RFP-HDFCs-Neo, ANGIO-PROTEOMIE, cAP-0008-NeoRFP, Boston, MA, USA) was used.

Techniques: Alamar Blue Assay, Negative Control, Cell Culture, Positive Control

Journal: Journal of Functional Biomaterials

Article Title: The Impact of the Methacrylation Process on the Usefulness of Chitosan as a Biomaterial Component for 3D Printing

doi: 10.3390/jfb15090251

Figure Lengend Snippet: Expression of selected genes in RFP-HDFCs-Neo cells after exposure to biomaterials compared to cells cultured under standard conditions (defined as 1-fold change). ( A ) Gene expression of cells cultured on the surface of biomaterials; ( B ) gene expression of cells in 3D-printed models. BCH: cells+ chitosan-based biomaterial; BCM: cells+ methacrylated chitosan-based biomaterial.

Article Snippet: The RFP Expressing Human Dermal Fibroblasts-Neonatal cell line (RFP-HDFCs-Neo, ANGIO-PROTEOMIE, cAP-0008-NeoRFP, Boston, MA, USA) was used.

Techniques: Expressing, Cell Culture, Gene Expression

Journal: BMC Cell Biology

Article Title: Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

doi: 10.1186/1471-2121-11-85

Figure Lengend Snippet: Organisation of the ASPM protein and its cellular distribution during mitosis . A . Structure of the 3477 amino acid human ASPM protein. The region corresponding to the microtubule-binding domain of Drosophila Asp is shown in white. The calponin homology domains (aa920-1261) are in light grey; ASNP repeats are dark grey boxes (aa316-342, 366-400); 81 IQ domains (aa1267-3225) are shown as vertical stripes; the armadillo repeat-like domain (aa3294-3327) is a black box; and the C -terminal region is depicted by diagonal stripes. The location of potential nuclear localization sequences are indicated by asterisks and the location of the peptides used to raise polyclonal antibodies, by dashed arrows. B-F . Analysis of ASPM distribution following immunostaining. HeLa cells were fixed and stained with antibodies specific for ASPM (green), anti-α-tubulin (red) and with DAPI (blue) to identify nuclei. Panels B-E utilised Anti-ASPM 216-1, whilst anti-ASPM 279-3 was used in panel F. B . ASPM is predominantly nuclear in interphase cells. Scale bar = 10 μm. C . ASPM is localised to the spindle poles during metaphase. Scale bar = 5 μm. D . A globular distribution of ASPM (green) is seen around the γ-tubulin (red) immunopositive core of metaphase HeLa cell spindle poles. DNA (DAPI staining) is shown in blue (confocal image). Scale bar = 2.5 μm. E . Single 0.5 μm confocal section through the centre of a telophase HeLa cell immunostained to reveal ASPM (green) and α-tubulin (red). In addition to broad spindle pole-associated labelling (arrowhead), ASPM also localizes to the minus ends of central spindle microtubules (arrow). Scale bar = 2.5 μm. F . A late telophase fibroblast immunostained with anti-ASPM 279-3 (green), anti-α-tubulin (red) and with DAPI (blue). ASPM is predominantly localized at the midzone of the central spindle. Scale bar = 10 μm. G . Immunoblotting of cell lysates of COS7 cells (lane 1), U2OS cells (lane 2), primary fibroblasts (lane 3) and HeLa cells (lane 4) with anti-ASPM 217-2 antibody. This identifies a protein of approximately 410 kDa in each lane. A blot stained with anti-β-actin is shown as loading control.

Article Snippet: Human neonatal dermal fibroblasts ( ASPM wt ) were obtained from Genlantis (San Diego, California).

Techniques: Binding Assay, Immunostaining, Staining, Western Blot, Control

Journal: BMC Cell Biology

Article Title: Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

doi: 10.1186/1471-2121-11-85

Figure Lengend Snippet: ASPM IVS25+1G > T mutation produces a novel splice variant with decreased efficiency for spindle pole localisation . A . Schematic of the ASPM protein showing the location of the ASPM IVS25+1G > T splice site mutation (filled arrows with an upper diamond). Domains of the protein are indicated as described in the legend to Figure 1. Location of epitopes of anti-ASPM antibodies are shown beneath the protein. B . Fibroblasts at metaphase immunostained using 216-1 anti-ASPM antibody (N-terminal) and the 279-3 anti-ASPM antibody, raised against a C-terminal peptide sequence. Note the reduced ASPM localization at the mitotic spindle poles of ASPM IVS25+1G > T cells. Scale bar = 10 μm. C . Chart representing integral intensity of ASPM 216-1 antibody staining at the spindle poles of ASPM wt and ASPM IVS25+1G > T fibroblast cells. Horizontal lines indicate the average integral intensity of the immunostaining. Standard error bars are present. ASPM IVS25+1G > T was at statistically significantly lower level (asterisks) at the spindle poles than ASPM wt when compared using a paired two tailed t-test. P = < 0000.1. D . Immunoblotting of fibroblast control (WT) and ASPM IVS25+1G > T (Patient) lysates with 217-2 anti-ASPM antibody and anti β-actin. Equivalent levels of stable ASPM protein are expressed by each cell culture, indicating the IVS25+1G > T mutation does not induce nonsense mediated decay. E . Sequence analysis of exon 25-exon 26 control ASPM wt and Patient ASPM IVS25+1G > T cDNA. The point of removal of the nine nucleotides due to the IVS25+1G > T mutation is indicated by an asterisk. F . IVS25+1G > T mutation removes the exon 25 splice donor site and instigates the utilisation of an in frame splice donor site nine nucleotides downstream. The resultant ASPM IVS25+1G > T protein is lacking just three amino acids (3326-3328).

Article Snippet: Human neonatal dermal fibroblasts ( ASPM wt ) were obtained from Genlantis (San Diego, California).

Techniques: Mutagenesis, Variant Assay, Sequencing, Staining, Immunostaining, Two Tailed Test, Western Blot, Control, Cell Culture