control human dermal fibroblast cell line  (ATCC)

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

Journal: Experimental Biology and Medicine

Article Title: N-acetyl-L-cysteine improves mitochondrial and oxidative defects in the acadian variant of fanconi syndrome

doi: 10.3389/ebm.2025.10448

Figure Lengend Snippet: Fibroblasts derived from Acadian variant of Fanconi syndrome (AVFS) patients have mitochondrial deficits and oxidative damage. (A) Immunoblotting and quantification of mitochondrial proteins in fibroblasts derived from control or AVFS patients showing lower levels of NDUFAF6 and the complex I subunit NDUFA9 but not of SDHA, UQRCRC2 and TOM20. (B) The enzymatic activity of complex I is decreased in AVFS fibroblasts (n = 9). (C) Fluorescence of the mitochondrial membrane potential-dependent TMRM is decreased in AVFS fibroblasts (n = 6). (D) Oxygen consumption rates (OCR) at different respiratory states indicating lower mitochondrial metabolism in AVFS fibroblasts (n = 3-4). (E–G) Levels of (E) 8-hydroxy-2′-deoxyguanosine (8-OHdG), (F) malondialdehyde (MDA) and (G) carbonyl, which indicate oxidative damage on DNA, lipid and protein, respectively, are increased in AVFS fibroblasts (n = 3). Data are presented as mean +- SEM. Data with different letters are statistically different, as measured by one way ANOVA followed by post-hoc Tukey test.

Article Snippet: Control fibroblasts were obtained from ATCC (ref. PCS-201-012).

Techniques: Derivative Assay, Variant Assay, Western Blot, Control, Activity Assay, Fluorescence, Membrane

Journal: Experimental Biology and Medicine

Article Title: N-acetyl-L-cysteine improves mitochondrial and oxidative defects in the acadian variant of fanconi syndrome

doi: 10.3389/ebm.2025.10448

Figure Lengend Snippet: Treatment with the antioxidant N-Acetyl-L-cysteine (NAC) reverses oxidative damage in fibroblasts derived from Acadian variant of Fanconi syndrome (AVFS) patients. (A) TMRM fluorescence in control fibroblasts do not change after treatment with NAC (1 mM, 5 days, n = 9). (B) MDA levels in control fibroblasts do not change after treatment with NAC (n = 9). (C) Representative immunoblotting (n = 3) of NDUFAF6 in control and AVFS fibroblasts with vehicle or NAC, showing that NAC does not rescue levels of NDUFAF6. (D) The enzymatic activity of complex I is partly rescued in AVFS fibroblasts upon treatment with NAC (n = 12). (E) TMRM fluorescence is partly rescued in AVFS fibroblasts treated with NAC (n = 6-7). (F) Oxygen consumption rates (OCR) at different respiratory states are partly rescued upon treatment with NAC (n = 3) (G–I) Levels of (G) 8-hydroxy-2′-deoxyguanosine (8-OHdG), (H) malondialdehyde (MDA) and (I) carbonyl, which indicate that oxidative damage on DNA, lipid and protein, respectively, are partly rescued in AVFS fibroblasts treated with NAC (n = 3). Data are presented as mean +- SEM. Data with different letters are statistically different, as measured by one way ANOVA followed by post-hoc Tukey test.

Article Snippet: Control fibroblasts were obtained from ATCC (ref. PCS-201-012).

Techniques: Derivative Assay, Variant Assay, Fluorescence, Control, Western Blot, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The DNase TREX1 is a substrate of the intramembrane protease SPP with implications for disease pathogenesis

doi: 10.1007/s00018-025-05645-5

Figure Lengend Snippet: SPP-dependent destabilization of TREX1 in AGS patients with homozygous T303P mutation. A Dermal fibroblasts from patients carrying the TREX1 303P mutation as well as two independent commercially obtained control fibroblast cell lines were monitored for their TREX1 levels by Western Blotting. B TREX1 mRNA abundance was quantified in the same cell lines by qPCR. Mean mRNA levels were normalized to those of control #1. N = 2, n = 3(patient #2), n = 4 (rest). One-Way ANOVA with Tukey’s post hoc test. ** p ≤ 0.01, *** p ≤ 0.001. C Patient fibroblasts were treated with 1 µM inhibitor X (InX) or DMSO as control for 24 h. TREX1 protein levels were finally compared to those from WT control lines by Western Blotting. D PBMCs were isolated from the two patients carrying the TREX1 T303P mutation as well as their mother (heterozygous carrier) or a non-related healthy volunteer. Cells were subsequently treated with DMSO or 1 µM inhibitor X (InX) for 24 h prior to cell lysis. Finally, cellular TREX1 levels were evaluated by Western Blotting

Article Snippet: Control human dermal adult fibroblasts were purchased from either ATCC (PCS-201-012) or Thermo Fisher Scientific (C0135C).

Techniques: Mutagenesis, Control, Western Blot, Isolation, Lysis

Journal: bioRxiv

Article Title: RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

doi: 10.1101/2024.04.03.587966

Figure Lengend Snippet: (A) RUNX1 expression rate in forearm skin biopsies of dcSSc ( N =49), lcSSc ( N =17), and healthy ( N =20) patients. (B) The expression rate of RUNX1 over the course of three years (data presented for 0, 6, 12, 24, and 36 months). (C) RUNX1 expression rate for healthy and SSc patients at early or late stages of disease at baseline. (D) Correlation between RUNX1 expression and mRSS skin score at baseline for both lcSSc (yellow) and dcSSc (red) ( N =66); early-stage patients are shown as a triangle and late-stage patients as a circle. (E) GSVA enrichment scores of main cellular signatures in healthy ( N =20), patients with RUNX1 high ( N =21) and RUNX1 low ( N =45). Hedge’s g effect size of RUNX1 high vs. RUNX1 low is presented in the graph. (F) Correlation between the TGF-β fibroblast and monocyte and myeloid cell signatures with RUNX1 .

Article Snippet: We analyzed a DNA microarray dataset previously generated by our lab, consisting of two independent SSc fibroblasts, one healthy control fibroblast isolated in parallel, and one normal human dermal (NHD) fibroblast cell lines obtained from ATCC.

Techniques: Expressing

Journal: bioRxiv

Article Title: RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

doi: 10.1101/2024.04.03.587966

Figure Lengend Snippet: (A) Schematic graph illustrating the timeline for the culture and TGF-β1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and Normal Human Dermal (NHD) fibroblast cells. (B) RUNX1 expression rate in samples treated with TGF-β1 (in red) vs. control for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the two 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-β1 treatment vs. the baseline. (E) Fold change expression of TGF-β1 and CBFB in TGF-β1-induced SSc fibroblasts treated with Ro5-3335 ( RUNX1 inhibitor), compared to control. (F) Proliferation curve of 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. SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. (Student’s t-test P- value: **0.001–0.01, ****<0.0001 in GraphPad Prism v9)

Article Snippet: We analyzed a DNA microarray dataset previously generated by our lab, consisting of two independent SSc fibroblasts, one healthy control fibroblast isolated in parallel, and one normal human dermal (NHD) fibroblast cell lines obtained from ATCC.

Techniques: Isolation, Expressing, Control, Positive Control

Journal: bioRxiv

Article Title: RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

doi: 10.1101/2024.04.03.587966

Figure Lengend Snippet: (A) THBS1 and RUNX1 expression levels in dcSSc skin biopsies of patients who were given two low doses (1 mg/kg) of fresolimumab at weeks 1 and 3 in yellow ( N =7); or a single high dose (5 mg/kg) of fresolimumab at week 1 in blue ( N =7). The mid-forearm skin biopsies were collected at baseline and again at weeks 3, 7, and 24. (B) The heatmap of genes in the TGF-β fibroblast cell signature for patients who received a high dose of fresolimumab at baseline and again 3 weeks after treatment ( N =7). (C) The expression of several genes including RUNX1 and TGF-β pathway biomarkers such as COMP , THBS1 , and FN1 .

Article Snippet: We analyzed a DNA microarray dataset previously generated by our lab, consisting of two independent SSc fibroblasts, one healthy control fibroblast isolated in parallel, and one normal human dermal (NHD) fibroblast cell lines obtained from ATCC.

Techniques: Expressing

Journal: bioRxiv

Article Title: RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

doi: 10.1101/2024.04.03.587966

Figure Lengend Snippet: (A) DNA methylation profile of 2D- and 3D-cultured fibroblasts isolated from dcSSc patients or healthy donors, created using Illumina’s Infinium Methylation EPIC array. Heatmap shows top 592 methylated CpG sites, with blue/yellow gradient of beta values. The bar-plot on top shows RUNX1 beta value that is labeled within the heatmap, showing that RUNX1 is hypomethylated in dcSSc samples. (B) Result of paired-wise differentially methylated CpGs and the number of significant CpGs in each group. (C) Pathway enrichment analysis of Reactome gene sets using top significant CpGs identified in (B) for each 2D and 3D culture. (D) The beta values of representative CpGs in RUNX1 locus in 2D and 3D SSc and healthy conditions. (E) RUNX1 locus on chromosome 21 and common CpG islands in green. The differentially methylated regions (DMRs) are identified between SSc and healthy samples are shown in red. The beta values corresponding to the CPGs at DMRs for SSc (in orange) and healthy (in green).

Article Snippet: We analyzed a DNA microarray dataset previously generated by our lab, consisting of two independent SSc fibroblasts, one healthy control fibroblast isolated in parallel, and one normal human dermal (NHD) fibroblast cell lines obtained from ATCC.

Techniques: DNA Methylation Assay, Cell Culture, Isolation, Methylation, Labeling

Journal: bioRxiv

Article Title: RUNX1 is Expressed in a Subpopulation of Dermal Fibroblasts and Higher RUNX1 Levels are Associated with the Severity of Systemic Sclerosis

doi: 10.1101/2024.04.03.587966

Figure Lengend Snippet: (A) UMAP projection of cell types from Tabib et al., 2021’s scRNA-seq of forearm skin biopsies (B) RUNX1 -normalized aggregate expression of 10 samples from healthy donors and 12 from dcSSc patients. (C) UMAP projection of 10 fibroblast subpopulations (clusters 0–9). Two fibroblast populations of 2 and 4 are marked, which are enriched SSc samples. (D) Feature plots of RUNX1 expression in healthy and SSc fibroblasts. Two RUNX1 -expressing fibroblast clusters are marked with their respective numbers. (E) The log-normalized expression rate of main differentially expressed genes between RUNX1 high - with RUNX1 low -expressing SSc fibroblasts. (F) Density plots of RUNX1 and major SSc-relevant genes within SSc fibroblast subpopulations. Arrows indicate the cluster 2 and 4 of SSc-specific subpopulations of fibroblasts.

Article Snippet: We analyzed a DNA microarray dataset previously generated by our lab, consisting of two independent SSc fibroblasts, one healthy control fibroblast isolated in parallel, and one normal human dermal (NHD) fibroblast cell lines obtained from ATCC.

Techniques: Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: ABE restores SLC17A5 c.115C sequence without creating indels in HDFs (A) DNA and respective protein sequences of SLC17A5 around locus c.115. (Top left) c.115C site (in green) and CGT coding for arginine at codon 39 of the SLC17A5 protein. (Top right) pathogenic variant SLC17A5 c.115C>T (in red), coding for cysteine at codon 39 of the SLC17A5 protein. (Middle) sgRNA design for HDR and ABE. The SLC17A5 c.115 site for the targeted cytosine-to-thymine transition is highlighted in red. Horizontal arrows indicate sequences of sgRNAs targeting the SLC17A5 c.115 site. Protospacer adjacent motifs (PAMs) are highlighted in color corresponding with the respective sgRNA: for HDR, in blue, and base editing, in orange. (Bottom) Sequence of single-stranded oligonucleotide (ssODN) used for HDR around SLC17A5 c .115C>T. PAM motifs are indicated in blue. A synonymous substitution to inactivate the PAM site ( SLC17A5 c.135G>A) is underlined. (B) Comparison of editing efficiency (green) and indel (gray) (%) between HDR and ABE in HDFs harboring the SLC17A5 c.115C>T variant (affected). (C) Temporal assessment of editing efficiency in HDFs following base editing from day zero to day 14 post-nucleofection. (D) Electropherograms of SLC17A5 sequence flanking c.115 in unaffected control, affected, HDR-treated, and ABE-treated HDFs, respectively. Shaded blue regions in the background of chromatograms represent Phred base calling quality scores. SLC17A5 c.115 site distinguished by arrows. (Top left) SLC17A5 c.115 unaffected control HDFs. Note the canonical ‘C’ nucleotide at position c.115. (Top right) Homozygous SLC17A5 c.115C>T variant HDFs. Note the mutated ‘T’ nucleotide at position c.115. (Bottom left) HDR-treated SLC17A5 c.115C>T HDFs. Multiple electropherogram peaks indicative of indels. (Bottom right) ABE-treated SLC17A5 c.115C>T affected HDFs. Note the dual peak for ‘T’ and ‘C’, indicating partial correction of the pathogenic ‘T’ to the canonical ‘C’ nucleotide at c.115. Bystander edit at c.114 is marked with an asterisk. Data generated from at least three independent experiments are shown as mean ± standard deviation. Editing efficiency (%) and indel (%) were analyzed using unpaired t tests. Temporal assessment was analyzed using one-way ANOVA with the Tukey post hoc test. ∗∗∗∗p < 0.0001.

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Sequencing, Variant Assay, Comparison, Control, Generated, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: ABE reduces markers of endo-lysosomal pathology and normalizes FSA levels in HDFs (A and C) LAMP1(A) and CD63 (C) staining in unaffected control (+/+), affected homozygous SLC17A5 c.115C>T (−/−), and ABE-treated SLC17A5 c.115C>T HDFs (ABE). Scale bar, 200 μm. (B and D) Quantification of LAMP1 (B) and CD63 (D) signal intensity in HDFs, measured in mius. The editing efficiency of the ABE-treated group used for staining corresponds with 53%. (E) Quantification of FSA in HDFs. Increased FSA levels in untreated affected HDFs (−/−) compared to unaffected control HDFs (+/+). Data generated from at least (n = 3) independent experiments are shown as mean ± standard deviation. All comparisons were analyzed using a one-way ANOVA with the Tukey post-hoc test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. See also Figures S1– S8 .

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Staining, Control, Generated, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: Targeted amplicon deep sequencing of putative off-target sites nominated in silico show no significant adenine deamination Putative off-target (OT) sites were determined by Cas-OFFinder using the c.115T 20-nucleotide query sequence. (A) OT site frequencies predicted by the Cas-OFFinder algorithm using a mismatch parameter of five or fewer nucleotides (5MM) compared with three or fewer nucleotides (3MM). (B) Predicted OT sites separated by genomic location type, with a majority of predicted OTs found to be in intergenic or intronic regions. (C) Heatmap (left) representing percentage of non-adenine base identities where adenine is expected, or indels (right), across in silico -nominated off-target sites after targeted amplicon deep sequencing and CRISPResso2 analysis. No significant differences are observed between ABE-treated SLC17A5 c.115C>T HDFs (ABE; bottom) and mock (top) samples. Associated OT sequences are listed at right, and adenines with the potential for deamination are in red (n = 3, unpaired Student t tests). See also Tables S2 and , Figures S9 and .

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Amplification, Sequencing, In Silico

Journal: Molecular Therapy. Nucleic Acids

Article Title: Base editing corrects the common Salla disease SLC17A5 c.115C>T variant

doi: 10.1016/j.omtn.2023.08.024

Figure Lengend Snippet: ABE restores Slc17a5 c.115C in a murine cell line (A) Human and murine sgRNA comparison. Murine and human sequences are identical, except a single nucleotide difference, highlighted in yellow. SLC17A5/Slc17a5 c.115C>T is in red. (B) Base-editing efficiency (green) and indel (gray) (%) MEFs harboring the SLC17A5 c.115C>T variant. (C) FSA quantification in MEFs. Increased FSA levels in untreated Slc17a5 c.115C>T MEFs (−/−) compared to unaffected control MEFs (+/+). ABE-treated Slc17a5 c.115C>T MEFs (ABE) demonstrate normalization of FSA. Data generated from at least (n = 3) independent experiments are shown as mean ± standard deviation. All comparisons were analyzed using a one-way ANOVA with the Tukey post hoc test. ∗∗∗∗p < 0.0001.

Article Snippet: For determination of LAMP1 and CD63 signal by fluorescence microscopy, unaffected control (HDFs, PCS-201-010; ATCC), affected SLC17A5 c.115C>T (GM08497), and ABE-treated SLC17A5 c.115C>T HDFs were grown on eight-chamber cell culture slides (CellTreat Scientific Products).

Techniques: Comparison, Variant Assay, Control, Generated, Standard Deviation