healthy control fibroblast lines  (ATCC)

Journal: Journal of Biomedical Science

Article Title: Modeling CLN3 Batten disease in astrocytes reveals alterations in mitochondria homeostasis, fatty acid metabolism and oxidative stress response

doi: 10.1186/s12929-026-01253-y

Figure Lengend Snippet: Generation and characterization of iPSC-derived astrocytes. A Schematic overview of astrocyte differentiation from patient-derived iPSCs. Key compounds used to drive differentiation toward a mature astrocyte phenotype are indicated: LIF (leukemia inhibitory factor), CHIR99021, SB431542, CoE, FGF2 (fibroblast growth factor 2), EGF (epidermal growth factor), and CNTF (ciliary neurotrophic factor). B qPCR analysis of astrocyte-specific markers (ALDHL1, GLAST, S100b, GFAP, Vimentin) in iPSC-derived cells. Expression of MAP2 (neuron marker) and OLIG2 (oligodendrocyte marker) was assessed to evaluate cell population purity. C Phase-contrast images and immunocytochemical validation of astrocyte marker expression. Vimentin and GFAP (green), S100β (red), and DAPI-stained nuclei (blue) are shown. D Quantification of CLN3 protein levels in control and CLN3 patient-derived iPSC and astrocytes via targeted mass spectrometry

Article Snippet: Healthy control fibroblast lines (two cell lines) were obtained from ATCC (cat. number PCS-201—012) and the Coriell Institute (cat. number AG05836).

Techniques: Derivative Assay, Expressing, Marker, Biomarker Discovery, Staining, Control, Mass Spectrometry

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Defective signaling and ISG induction downstream of IFNAR2 p.Ser53Pro. (A) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNβ 100 IU/ml for 5, 15, or 30 min. Whole-cell lysates were harvested for Western blotting for the visualization of pSTAT1, STAT1, and GAPDH, which was used as the loading control. Not repeated due to limited patient material. (B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were treated with IFNβ 100 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and the visualization of IFNAR2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (C) Primary IFNAR2 −/− dermal fibroblasts reconstituted with lentiviruses expressing GFP, WT, or p.Ser53Pro IFNAR2 were treated with IFNα2b or IFNγ 1,000 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and visualization of pJAK1, JAK1, pSTAT2, STAT2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (D) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNβ 100 IU/ml for 6 h. Total RNA was purified for RT-qPCR of IFIT1 and IRF7 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001, ns, non-significant; two-way ANOVA with Šidák’s test for multiple comparisons). (E) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the mother of P1 (Het) were treated with IFNβ 100 IU/ml for 6–24 h. Total RNA was purified for RT-qPCR of MX1 and IFIT1 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Dunnett’s test for multiple comparisons). (F) Primary dermal fibroblasts from P1 or a healthy control (C1) were treated with IFNα2b 1,000 IU/ml overnight. Whole-cell lysates were prepared for immunoblotting and visualization of MX1, USP18, RSAD2, ISG15, and α-tubulin (ATUB) as the loading control. One representative immunoblot of n = 3 independent experiments is shown. Source data are available for this figure: .

Article Snippet: Healthy control human dermal fibroblast lines were obtained from PromoCell or existing stocks.

Techniques: Western Blot, Expressing, Purification, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: In vitro functional modelling of the IFNAR2 p.Ser53Pro mutant. (A) EBV-transformed B cells from P2 and a healthy control (C1) were treated with IFNα2b 1,000 IU/ml for 30 min. Whole cell lysates were harvested for immunoblotting and visualization of STAT1, pSTAT1 and GAPDH as loading control. Representative of n = 2 repeat experiments. (B) Graphical summary of experimental strategy. IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control. (C) IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control and protein lysates prepared for immunoblotting for IFNAR2, GFP, and GAPDH loading control. Representative of n = 3 independent experiments. (D) IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control. After overnight stimulation with IFNα2b or IFNγ (1,000 IU/ml) protein lysates were prepared for immunoblotting for the ISG products MX1, USP18, RSAD2 and ISG15 alongside GAPDH loading control. Representative of n = 3 independent experiments. (E) Primary IFNAR2-deficient fibroblasts, stably reconstituted with empty vector, WT IFNAR2 or p.Ser53Pro IFNAR2 , were pre-treated with IFNα2b or IFNγ (1,000 IU/ml) overnight, prior to infection with HSV1 (17 + strain, MOI 0.01). At 48 h after infection, lysates were prepared for immunoblotting for HSV gD, alongside MX1, RSAD2, ISG15 and GAPDH as loading control. Representative of n = 3 independent experiments. (F) Primary IFNAR2-deficient fibroblasts, stably reconstituted with empty vector, WT IFNAR2 or p.Ser53Pro IFNAR2 , were pre-treated with IFNα2b or IFNγ (1,000 IU/ml) overnight, before infection with a cytopathic dose of EMCV. At 24 h after infection, cell viability was assessed in an imaging based live cell viability assay (mean ± SD of n = 3 independent experiments, one-way ANOVA with Šidák’s multiple comparisons test; ****, P < 0.0001; ns, non-significant). Source data are available for this figure: .

Article Snippet: Healthy control human dermal fibroblast lines were obtained from PromoCell or existing stocks.

Techniques: In Vitro, Functional Assay, Mutagenesis, Transformation Assay, Western Blot, Construct, Plasmid Preparation, Stable Transfection, Infection, Imaging, Viability Assay

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Impaired viral control in patient fibroblasts bearing homozygous IFNAR2 p.Ser53Pro. (A and B) SV40-immortalized dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were pretreated with IFNβ 100 IU/ml for 24 h before infection with HSV1 (KOS strain) at an MOI of 0.001. (A) At 24, 48, and 72 hpi, supernatants were sampled and titrated for TCID50 (geometric mean ± SD of n = 3 independent repeats; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA with Tukey’s test for multiple comparisons). (B) At 72 hpi, the cells were lysed for immunoblotting of whole-cell lysates for the HSV1 protein VP5 and GAPDH as the loading control. One representative immunoblot of n = 3 independent repeat experiments is shown. (C) SV40-immortalized dermal fibroblasts from P1 and a healthy control were pretreated with IFNβ 100 IU/ml for 24 h before infection with cell-free VZV at an MOI of 1. At 48 hpi, total RNA was harvested for RT-qPCR, evaluating the levels of VZV immediate early ORF63 and late ORF40 transcripts, respectively, relative to GAPDH (mean ± SD of n = 3 independent repeats; *, P < 0.05; ***, P < 0.001, two-way ANOVA with Šidák’s test for multiple comparisons). (D) SV40-immortalized dermal fibroblasts from P1 and one healthy control were pretreated with IFNβ 100 IU/ml for 24 h before infection with MeV (Edmonston strain, MOI = 0.00083). At 96 hpi, supernatants were harvested and titrated for TCID50 (geometric mean ± SD of n = 3 independent repeats; ****, P < 0.0001, two-way ANOVA with Tukey’s test for multiple comparisons). (E and F) Primary dermal fibroblasts from P1, a healthy control (C1), the heterozygous mother of P1 (Het) and a known IFNAR2-deficient patient ( IFNAR2 −/− ) were pretreated with IFNα2b 1,000 IU/ml for 16 h before infection with MuV at an MOI 0.1 (MuV, Enders strain). At 72 hpi, (E) viability was assessed in an imaging based live cell viability assay (mean ± SD of n = 3 independent repeats; **, P < 0.01; ****, P < 0.0001, two-way ANOVA with Dunnett’s test for multiple comparisons) and (F) whole cell lysates were prepared for immunoblotting and visualization of MuV nucleoprotein (NP) and GAPDH as loading control. One representative immunoblot of n = 3 independent repeat experiments is shown. (G) Primary dermal fibroblasts from P1 and a healthy control (C1) were pretreated with IFNα2b 1,000 IU/ml for 16 h before infection with a cytopathic dose of EMCV. At 24 hpi, cell viability was assessed in an imaging based live cell viability assay (mean ± SD of n = 3 independent repeats; ****, P < 0.0001, two-way ANOVA with Dunnett’s test for multiple comparisons). Source data are available for this figure: .

Article Snippet: Healthy control human dermal fibroblast lines were obtained from PromoCell or existing stocks.

Techniques: Infection, Western Blot, Quantitative RT-PCR, Imaging, Viability Assay

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Defective processing of p.Ser53Pro IFNAR2 through the secretory pathway. (A) Primary dermal fibroblasts from P1, healthy controls (C1–3), and the heterozygous mother of P1 (Het) were lysed for immunoblotting of whole cell lysates for IFNAR2 protein and α-tubulin (ATUB) as loading control. Densitometry analysis of IFNAR2 expression relative to loading control (mean ± SD of n = 3 independent experiments; *, P < 0.05; **, P < 0.01; ****, P < 0.0001, Welch’s one-way ANOVA with Dunnett’s test for multiple comparisons). (B) Total RNA from primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) was purified for RT-qPCR evaluating IFNAR2 mRNA levels relative to TBP (mean ± SD of n = 3 independent experiments; *, P < 0.05; **, P < 0.01, one-way ANOVA with Tukey’s test for multiple comparisons). (C) Model of change to IFNAR2 protein entropy from the p.Ser53Pro substitution, prepared using DynaMUT. Red color indicates a gain of flexibility, blue a gain of stability. (D) HEK 293 FT cells were transfected with HA-tagged expression constructs encoding WT or p.Ser53Pro IFNAR2 . 24 h after transfection, the cells were treated with CHX at the indicated concentrations or DMSO vehicle control, for a further 24 h, before whole-cell lysates were prepared for immunoblotting for IFNAR2, and α-tubulin (ATUB) as loading control. Expression of IFNAR2 protein relative to loading control was assessed by densitometry analysis and expressed as a proportion of the DMSO-treated control. Displayed is a representative immunoblot of two independent experiments. (E) Left: HEK293FT cells were transfected with lentiviral expression constructs expressing IFNAR2 WT or IFNAR2 p.Ser53Pro or empty vector. Whole cell lysates were prepared and treated with PNGase F before immunoblotting for IFNAR2 and GAPDH as loading control. Displayed is a representative immunoblot of n = 3 independent experiments. (E) Right: Primary dermal fibroblasts of a healthy control were stably transduced with a lentiviral vector encoding IFNAR2 WT or IFNAR2 p.Ser53Pro. Whole cell protein lysates were harvested and treated with O-glycosidase and neuraminidase before immunoblotting for IFNAR2 and GAPDH as loading control. Displayed is a representative immunoblot of n = 2 independent experiments. (F) HEK293FT cells were transfected with HA-tagged expression constructs encoding WT or p.Ser53Pro IFNAR2 . 24 h after transfection cells were fixed, immunostained, and imaged by confocal microscopy for expression of the Golgi marker RCAS1 (left) or the ER marker calnexin (right) alongside IFNAR2. Scale bars represent 10 μm. Shown are the results of correlation analysis of IFNAR2 and the relevant organelle marker in individual cells (mean ± SD of n = 9 WT and n = 12 S53P cells analyzed; ***, P < 0.001, t test). Representative of n = 2 repeat experiments. (G) HEK293FT cells were transfected with HA-tagged expression constructs encoding WT or p.Ser53Pro IFNAR2 . 24 h after transfection cell surface expression of IFNAR2 was assessed by flow cytometry. Representative of n = 2 repeat experiments. Source data are available for this figure: .

Article Snippet: Healthy control human dermal fibroblast lines were obtained from PromoCell or existing stocks.

Techniques: Western Blot, Expressing, Purification, Quantitative RT-PCR, Transfection, Construct, Plasmid Preparation, Stable Transfection, Transduction, Confocal Microscopy, Marker, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Reconstitution of patient fibroblasts with WT IFNAR2 restores IFNAR2 signaling, ISG induction and viral control. (A and B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were transduced with lentiviral vectors encoding EGFP , IFNAR2 WT, or IFNAR2 p.Ser53Pro. 2 d after transduction, cells were pretreated with IFNβ 100 IU/ml for either (A) 30 min before being lysed and harvested for whole cell lysates for immunoblotting or (B) 6 h before being lysed and harvested for total RNA for RT-qPCR of MX1 and IFIT1 relative to GAPDH (mean ± SD of n = 3 independent repeats; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons). For A, one representative immunoblot of three independent experiments is shown. (C and D) SV40-immortalized dermal fibroblasts from P1 and a healthy control were transduced with lentiviral vectors encoding EGFP or IFNAR2 WT. 2 d after transduction, cells were pretreated with IFNβ 100 IU/ml for 24 h before infection with HSV1 (KOS strain) at an MOI of 1. At 24 h p.i., supernatants were harvested and titrated for (C) TCID50 (geometric mean ± SD of n = 3 independent replicates; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons) and the cells were lysed for Western blotting for the HSV1 protein VP5, IFNAR2, and GAPDH as loading control (D). Source data are available for this figure: .

Article Snippet: Healthy control human dermal fibroblast lines were obtained from PromoCell or existing stocks.

Techniques: Transduction, Western Blot, Quantitative RT-PCR, Infection