Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: DNA damage is a hallmark of commercial primary IPF fibroblasts in vitro , but not Cdkn2a /p16 ink4a expression or a fibrotic secretome . ( A ) Primary NHLF and IPF cells ( n = 3 different donors, each with 2 technical replicates) were cultured at the same density overnight on standard tissue culture plates in low-serum growth medium and were subsequently fixed. Immunocytochemistry was performed to fluorescently label nuclei (blue) and p21 Waf1/Cip1 (orange). Images were acquired with a 40X water objective using an Operetta High Content Screening instrument and intensity of nuclear p21 Waf1/Cip1 was quantified and calculated as a positive percentage of each population. ( B ) NHLF and IPF cells were treated and imaged as in ( A ), and immunocytochemistry was performed to fluorescently label nuclei (blue) and DNA damage via nuclear γH2A.X (Ser139) foci (orange). The number of nuclear foci per cell was quantified and cells with one or more were reported as positive. ( C ) Following overnight culture in standard tissue culture plates, NHLF and IPF cells were lysed, and qPCR was performed. Expression of senescence and fibrosis-related matrix and secreted factor genes was assessed, and data were normalized to β2m housekeeper expression using the 2 −ΔΔCt method versus NHLF cells. ( D ) Following overnight culture as in ( C ), cell culture supernatants were collected and assayed for determination of the concentration of common fibrosis-related secreted proteins by MSD kits. Statistical analysis was performed using an unpaired t -test ( A , B ) or a two-way ANOVA with a Bonferroni post-test ( C , D ) in GraphPad Prism: * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent standard error of the mean (SEM).

Article Snippet: For experiments, NHLFs and IPF fibroblasts were cultured in starve medium, containing 0.5% fetal bovine serum (Thermo Fisher Scientific) in the fibroblast basal medium (ATCC), 1% penicillin-streptomycin, and no additives from the low-serum growth kit.

Techniques: In Vitro, Expressing, Cell Culture, Immunocytochemistry, High Content Screening, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: Secreted factors from senescent alveolar epithelial cells potentiate pro-fibrotic gene expression and protein secretion more robustly than direct damage of fibroblasts. ( A ) Schematic summarizing the experimental protocol of direct damage of NHLFs with bleomycin or indirect treatment of NHLFs by transferring conditioned medium from bleomycin-treated AECs. Image created with https://www.biorender.com/ . ( B ) Treated or untreated NHLF cells were lysed, and qPCR gene expression analysis was performed. Expression of senescence and fibrosis-related matrix and secreted factor genes was assessed, and data were normalized to β2m housekeeper expression using the 2 −ΔΔCt method. ( C ) Senescent or non-senescent AEC conditioned medium or blank AEC medium was diluted 1:3 in fibroblast medium and incubated on NHLFs for 5 days. Cells were lysed for gene expression analysis of senescence and fibrosis-related genes. ( D ) Supernatants were collected from the treatments described in C and assayed for fibrosis-related secreted proteins by MSD kits. Data are representative of three independent experiments accounting for 3 biological donors of AECs and 2 donors of NHLFs. Statistical analysis was performed using a two-way ANOVA and a Dunnett’s Multiple Comparisons post-test vs. control conditions in GraphPad Prism: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant. Error bars represent SEM of n = 2 technical replicates.

Article Snippet: For experiments, NHLFs and IPF fibroblasts were cultured in starve medium, containing 0.5% fetal bovine serum (Thermo Fisher Scientific) in the fibroblast basal medium (ATCC), 1% penicillin-streptomycin, and no additives from the low-serum growth kit.

Techniques: Gene Expression, Transferring, Expressing, Incubation, Control

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: Senolytic tool agent Navitoclax and AD80 attenuate senescent AEC CM-mediated collagen secretion. Conditioned medium with 2.5 μM Navitoclax and 200 nM Nintedanib, ABT-317, ruxolitinib, or AD80 AEC treatment conditions from were diluted 1:3 in fibroblast starve medium and transferred to NHLFs for 5 days. Supernatants were assayed for collagen 1 with MSD kits. Data are presented as a percentage of the DMSO control in the matched treatment condition. Statistical analysis was performed using a two-way ANOVA and a Dunnett’s multiple comparisons test in GraphPad Prism versus each group’s DMSO control: * p < 0.05 and **** p < 0.0001. Data is representative of three independent experiments accounting for 3 biological donors of AECs and 2 donors of NHLFs. Error bars represent SEM of n = 5 (DMSO controls) or n = 2 (treatments) technical replicates.

Article Snippet: For experiments, NHLFs and IPF fibroblasts were cultured in starve medium, containing 0.5% fetal bovine serum (Thermo Fisher Scientific) in the fibroblast basal medium (ATCC), 1% penicillin-streptomycin, and no additives from the low-serum growth kit.

Techniques: Control

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: Summary of in vitro and ex vivo systems to mimic alveolar epithelial and fibroblast damage and dysfunction in the IPF lung. While IPF fibroblasts and bleomycin treated NHLFs exhibit some hallmarks of senescence, these cells do not appear to demonstrate a phenotype of Cdkn2a /p16 ink4a expression, ECM deposition, or secretion of fibrotic mediators such as TIMP1. Rather, the SASP of senescent, aberrant epithelial cells drives a fibrotic phenotype in NHLFs that is consistent with progressive fibrosis. Development of senolytic agents presents an opportunity for therapeutic impact early in disease pathogenesis and with an orthogonal mechanism than the fibroblast targeting standard of care, Nintedanib. Image created with https://www.biorender.com/ .

Article Snippet: For experiments, NHLFs and IPF fibroblasts were cultured in starve medium, containing 0.5% fetal bovine serum (Thermo Fisher Scientific) in the fibroblast basal medium (ATCC), 1% penicillin-streptomycin, and no additives from the low-serum growth kit.

Techniques: In Vitro, Ex Vivo, Expressing

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control

Journal: Frontiers in Physiology

Article Title: Thiamet-G facilitates reparative dentin formation via modulating O-GlcNAcylation and inflammation

doi: 10.3389/fphys.2025.1739168

Figure Lengend Snippet: Effect of Thiamet-G on human dental pulp stem cells (hDPSCs). (A) Experimental design for MTS assay to examine cell viability and proliferation. (B) MTS assay shows that Thiamet-G treatment facilitates dose-dependent hDPSC proliferation. (C) Experimental design for ALP activity assay. (D) Thiamet-G treatment significantly induces ALP activity in hDPSCs compared to the control, particularly after 14 days (E) Thiamet-G treatment increases the expression of reparative dentin-related markers, including Alp, Bmp2, Bsp, Dspp, Opn, Gsk3β, and RUNX2, compared to the control. Ns non-significant, * p < 0.03, ** p < 0.002, *** p < 0.0002, and **** p < 0.0001. NC negative control, GM growth medium, OM osteogenic medium.

Article Snippet: After 24 h, the media was changed with osteogenic media (Alfa-MEM with 1% penicillin-streptomycin, 5% fetal bovine serum, 50 μM L-ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone) in presence or absence of Thiamet-G (S7213, Selleckchem.com ) with various concentrations.

Techniques: MTS Assay, ALP Activity Assay, Activity Assay, Control, Expressing, Negative Control

Journal: Stem Cell Research & Therapy

Article Title: Significant improvement of bone marrow-derived MSC expansion from MDS patients by defined xeno-free medium

doi: 10.1186/s13287-023-03386-5

Figure Lengend Snippet: Immunophenotypic characterization. A Surface marker expression on cells freshly thawed or expanded over 2 passages with FBS or XF media. The cells are negative for hematopoietic lineage (lin) markers (CD45, CD34, CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a) and positive for mesenchymal markers (CD73, CD90, CD105, CD146 and CD271) B Flow cytometry plots showing the gating strategy with dead cell and lineage exclusion. C Representative histograms of thawed or expanded MSC stained for mesenchymal markers CD73, CD90, CD105, CD146 and CD271 (gray histograms). White histograms showing the respective unstained control

Article Snippet: To compare growth kinetics between healthy and MDS MSCs 330 MSCs/cm 2 freshly thawed samples were washed two-times in the target medium and were cultured with StemMACS human MSC Expansion Media (FBS) or StemMACS human MSC Expansion Media XF (Miltenyi Biotec, Bergisch Gladbach, Germany) for 12 days.

Techniques: Marker, Expressing, Flow Cytometry, Staining, Control