normal human dermal fibroblast nhdf cell line  (PromoCell)

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: International Microbiology

Article Title: Myconanotechnology: evaluation of Ag-Cu bimetallic nanoparticles synthesized by Ganoderma aff. australe against pathogens and cancer cells

doi: 10.1007/s10123-026-00793-5

Figure Lengend Snippet: Cytotoxic activity of Ganoderma aff. australe aqueous extract and Ag/Cu nanoparticles against cancer and non-cancerous cell lines evaluated by MTT assay. Cells (4,500 cells/well) were seeded in 96-well plates and allowed to adhere for 24 h before treatment. Concentrations tested were based on the amount of aqueous extract used to synthesize nanoparticles (see Table 1 ). All treatments were incubated with cells for 72 h at 37 °C with 5% CO₂. After incubation, cells were rinsed with PBS and incubated with 10 µl MTT solution (5 mg/ml) for 4 h, followed by addition of 100 µl DMSO. Absorbance was measured at 570 nm. Bar graphs show cell viability (expressed as IC₅₀ in mg/ml equivalent of extract) for five cancer cell lines and two non-cancerous control lines. Cancer cell lines: Caco-2 (colon cancer, ATCC HTB-37), HT-29 (colon cancer, ATCC HTN-38), MCF7 (breast cancer, ATCC HTB-22), A-172 (glioblastoma, ATCC CRL-1620), and U-87 MG (glioblastoma, ATCC HTB-14). Non-cancerous control lines: HDFn (human dermal fibroblasts, ATCC PCS-201-010) and Detroit 551 (normal skin fibroblasts, ATCC CCL-110). All cell lines were cultured in DMEM/F12 medium supplemented with 10% FBS, 1% antibiotic-antimycotic, 1% glutamine, 1% nonessential amino acids, and 1% sodium pyruvate. A Aqueous extract (0.5 g/50 ml) showing moderate cytotoxic activity with IC₅₀ values ranging from 1.61 ± 0.35 mg/ml (Caco-2) to 5.78 ± 1.48 mg/ml (Detroit 551), demonstrating baseline bioactivity of fungal metabolites. B M2-3-3 nanoparticles (2 ml extract + 3 ml AgNO₃ + 3 ml CuSO₄) exhibiting the highest cytotoxic efficacy across all cancer cell lines, with particularly remarkable activity against glioblastoma lines A-172 (IC₅₀: 0.26 ± 0.09 mg/ml) and U-87 MG (IC₅₀: 0.31 ± 0.12 mg/ml), and colorectal cancer lines Caco-2 (IC₅₀: 0.39 ± 0.12 mg/ml) and HT-29 (IC₅₀: 0.58 ± 0.28 mg/ml). Critically, M2-3-3 showed selective cytotoxicity with significantly higher IC₅₀ values in non-cancerous lines HDFn (2.87 ± 0.64 mg/ml) and Detroit 551 (3.45 ± 0.89 mg/ml), indicating preferential toxicity toward cancer cells. C M3-2-2 nanoparticles (3 ml extract + 2 ml AgNO₃ + 2 ml CuSO₄) demonstrating intermediate cytotoxic activity with IC₅₀ values consistently higher than M2-3-3 but lower than M5-3-3 across all cancer cell lines. D M5-3-3 nanoparticles (5 ml extract + 3 ml AgNO₃ + 3 ml CuSO₄) showing the lowest cytotoxic activity among the three nanoformulations, though still superior to the crude extract. Data represent mean ± SD of three independent experiments performed in triplicate. Statistical analysis performed using one-way ANOVA followed by Tukey’s HSD post-hoc test. Asterisks indicate significant differences: * p ≤ 0.050, ** p ≤ 0.010, *** p ≤ 0.001. The hierarchical efficacy pattern (M2-3-3 > M3-2-2 > M5-3-3 > crude extract) demonstrates successful nanotechnological enhancement of anticancer properties and establishes M2-3-3 as the optimal formulation with superior therapeutic selectivity. The exceptional sensitivity of glioblastoma cell lines suggests potential application in treating brain cancers, which are notoriously difficult to treat due to blood-brain barrier penetration challenges. Complete IC₅₀ values and statistical comparisons are provided in Table 4

Article Snippet: Fibroblast cell lines HDFn and Detroit 551 (ATCC, PCS-201-010 and CCL-110, respectively) were used to control non-cancerous cells.

Techniques: Activity Assay, MTT Assay, Incubation, Control, Cell Culture, Formulation

Journal: Antioxidants

Article Title: Bioenergetic Signatures of DLD Deficiency: Dissecting PDHc- and α-KGDHc-Linked Defects

doi: 10.3390/antiox15010019

Figure Lengend Snippet: Mitochondrial respiration is impaired in fibroblasts derived from patients with DLD deficiency. Mitochondrial oxygen consumption was assessed in controls (Ctrl1 and Ctrl2) and patient (Pt1–Pt6) fibroblasts using high-resolution respirometry (Oroboros O2k). ( A ) Routine respiration; ( B ) maximal respiration calculated as the difference between FCCP-stimulated and α-chaconine–permeabilized rates; ( C ) complex I-linked respiration (NADH-linked respiration, N-pathway), calculated as the difference between ADP and α-chaconine; ( D ) complex II-linked respiration (NS-pathway) calculated as the difference between respiration after rotenone and α-chaconine addition; ( E ) effect of complex I inhibition, calculated as the difference between FCCP-stimulated and rotenone-inhibited respiration; and ( F ) complex I/complex II respiration ratio (complex I-linked activity divided by complex II-linked activity). Each open circle represents an independent experimental run (N = 4–8 repeats per sample). All data were normalized to cell number. Statistical analysis was performed using the Mann–Whitney U test. * p < 0.05 vs. Ctrl1; numerical p values (0.05 < p < 0.1) are indicated on the plots. Abbreviations: Ctrl, control; Pt, patient.

Article Snippet: Dermal fibroblast primary cell lines from six genetically confirmed patients with DLD deficiency were obtained from the Pediatric Metabolic Disease Unit, Sheba Medical Center (IRB# SMC-21-8644, Figure 1, Table 1, and ), as well as two control cell lines: a control human dermal fibroblast cell line was purchased from ATCC (PCS-201-012; Ctrl 1, Manassas, VA, USA), and a primary cell line from a 39-year-old healthy male (Ctrl 2).

Techniques: Derivative Assay, Inhibition, Activity Assay, MANN-WHITNEY, Control