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: Medicina

Article Title: Evaluation of TAM Receptor Targeting in Pathophysiology of Idiopathic Pulmonary Fibrosis

doi: 10.3390/medicina61101837

Figure Lengend Snippet: Relative mRNA expression levels of Axl, Tyro3, and Gas6 genes in IPF FBs and HPFs quantified by RT-qPCR. No Mer expression was detected in both fibroblast types. TBP gene was used as housekeeping gene. N = 4.

Article Snippet: Control human pulmonary fibroblasts (HPFs, C12360 , PromoCell, St. Louis, MO, USA) were cultured in 25 mM glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin solution, and cells were used between passages 7 and 15.

Techniques: Expressing, Quantitative RT-PCR

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: JCI Insight

Article Title: A toxic gain-of-function variant in MAPK8IP3 provides insights into JIP3 cellular roles

doi: 10.1172/jci.insight.187199

Figure Lengend Snippet: ( A ) Cells (2,000 cells/well) were seeded in 96 wells for 3 days. Cell proliferation was measured by MTT assay (left). Apoptosis induction was measured by caspase-3/7 activity (right). All of results were normalized to healthy patient-derived fibroblasts (control cells, 2,936). n = 5, * P < 0.05, ** P < 0.01. ( B ) JIP3, JIP3, p-JNK, and total JNK proteins levels were measured by Western blot and normalized to GAPDH. ( C ) Trafficking of Cy3-transferrin along with microtubules in cells was detected with a Keyence microscope. Microtubules were stained by anti-tubulin antibody. mTOCs were marked with white dashed lines. Zoomed-in views were provided for the regions within the frames, with white arrows indicating representative colocalization sites. Scale bar: 50 μm; original magnification, ×63. ( D ) Cell imaging of either patient-derived fibroblasts or control fibroblasts incubated with 1 μM Cy3-transferrin for different time points. Colocalization of Cy3-transferrin and Rab7 (late endosome [LE] marker) was observed with a Keyence microscope. Zoomed-in views were provided for the regions within the frames, with white arrows indicating representative colocalization sites. Scale bar: 50 μm. ( E ) Cell imaging of either YFP-TLR4–transfected patient fibroblasts or healthy control fibroblasts incubated with 10 μg/mL LPS for different time points. Colocalization of activated YFP-TLR4 and Rab7 observed with a Keyence BZ-X800 microscope. Nuclei were marked with white dashed lines. Scale bar: 50 μm. ( F ) Cell imaging of lysosomes (LAMP1, a lysosome marker) either in patient-derived fibroblasts or healthy control fibroblasts observed by a Keyence microscope. Average distance between lysosome cluster to the center of nucleus in cells was measured by BZ-X800 analyzer software with the 3D model analysis function. Scale bar: 10 μm. Control cells, n = 85; patient cells, n =109. Unpaired t test.

Article Snippet: Three human dermal control fibroblasts (GM02936, GM03529 and GM03440) were purchased from Coriell Institute for Medical Research and cultured in DMEM with 10% FBS.

Techniques: MTT Assay, Activity Assay, Derivative Assay, Control, Western Blot, Microscopy, Staining, Imaging, Incubation, Marker, Transfection, Software

Journal: JCI Insight

Article Title: A toxic gain-of-function variant in MAPK8IP3 provides insights into JIP3 cellular roles

doi: 10.1172/jci.insight.187199

Figure Lengend Snippet: ( A ) Dopamine receptor 1 and 2 (D1 and D2) and p-AKT proteins levels in fibroblasts measured by Western blot. GAPDH was used as a loading control. Either healthy fibroblasts or patient fibroblasts were seeded in 96 wells (10,000 cells/well) and treated with 1 μM ( B ) D1-selective agonist SKF 81297 and ( C ) D2-selective agonist ropinirole for different time points. cAMP levels were measured with a cAMP-Glo Assay (Promega). Data were normalized to untreated control (UTC) of cells at time 0. n = 3. Either healthy individual–derived fibroblasts or patient-derived fibroblasts were seeded in 96 wells (8,000 cells/well) and treated by different concentrations of ( D ) D1-selective agonist SKF 81297 and ( E ) D2-selective agonist ropinirole for 30 minutes. cAMP levels were measured by cAMP-Glo Assay (Promega). Data were normalized to UTC of cells. n = 3. ( F ) Fibroblasts were treated with 1 μM D2-selective antagonist domperidone (Dom) for 5 minutes before addition of 1 μM D1-selective agonist SKF 81297 (SKF) for 30 minutes. Data were normalized to UTC of cells. n = 3. * P < 0.05. ( G ) Fibroblasts were treated with different concentrations of D2-selective antagonist domperidone (Dom) for 30 minutes. cAMP levels were measured by cAMP-Glo Assay (Promega). Data were normalized to UTC of cells. n = 3. ( H ) Fibroblasts were treated with 1 μM β1-AR–selective antagonist bisoprolol, β2-AR–selective antagonist ICH118551, and D1-selective antagonist SCH23390 for 30 minutes. cAMP levels were measured by cAMP-Glo Assay (Promega). Data were normalized to UTC of cells. n = 3. * P < 0.05, ** P < 0.01.

Article Snippet: Three human dermal control fibroblasts (GM02936, GM03529 and GM03440) were purchased from Coriell Institute for Medical Research and cultured in DMEM with 10% FBS.

Techniques: Western Blot, Control, Glo Assay, Derivative Assay

Journal: JCI Insight

Article Title: A toxic gain-of-function variant in MAPK8IP3 provides insights into JIP3 cellular roles

doi: 10.1172/jci.insight.187199

Figure Lengend Snippet: Healthy individual–derived (control) fibroblasts from GM03440 and patient-derived fibroblasts (5,000 cells/well) were electroporated with different concentrations of non-allele-selective ASO3 targeting MAPK8IP3 (JIP3) and incubated in 96 wells with normal culture medium for different time points for different experiments. Scrabble PS-ASO 676630 (5 μM) was used as a control ASO. ( A ) After 72-hour incubation, cell proliferations were measured by MTT assay and normalized to UTC of control cells. n = 3, * P < 0.05, ** P < 0.01. ( B ) After 48-hour incubation, RNA was extracted by GITC assay, and MT-MAPK8IP3 mRNA levels were measured using q-PCR. Data were normalized to UTC of control cells. n = 3, ** P < 0.01. ( C ) WT MAPK8IP3 mRNA levels were measured using q-PCR. Data were normalized to UTC of control cells. n = 3, ** P < 0.01. ( D ) SPAG9 (JIP4) mRNA levels were measured using q-PCR. Data were normalized to UTC of control cells. n = 3, ** P < 0.01. ( E ) Cell proteins were harvested after 72-hour incubation of ASOs with different concentrations. JIP3, JIP4, p-JNK, and total JNK were measured by Western blot. GAPDH was used as a loading control. ( F ) 10 μM non-allele-selective ASO3 was used to treat mutations in patient iPSC neurons for 72 hours, and then 1 μM D1 agonist (SKF 81297) was added to each test group with different incubation time points. cAMP levels were measured by cAMP-Glo Assay (Promega). Data were normalized to UTC of cells. n = 3, * P < 0.05. ( G ) After 48-hour ASO treatment, cell imaging of either patient fibroblasts or healthy control fibroblasts incubated with 1 μM Cy3-transferrin for different time points. Colocalization of Cy3-transferrin and Rab7 (late endosome [LE] marker) was observed with a Keyence BZ-X800 microscope. Scale bar: 20 μm.

Article Snippet: Three human dermal control fibroblasts (GM02936, GM03529 and GM03440) were purchased from Coriell Institute for Medical Research and cultured in DMEM with 10% FBS.

Techniques: Derivative Assay, Control, Incubation, MTT Assay, Western Blot, Glo Assay, Imaging, Marker, Microscopy

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