Journal: Journal of Human Immunity

Article Title: Human ISG15 deficiency unveils impaired healing of ulcerations via type I interferon–mediated fibrosis

doi: 10.70962/jhi.20250011

Figure Lengend Snippet: Identification of a patient with a novel ISG15 mutation. (A) Family includes two affected children carrying a homozygous variant (c.463insC) on ISG15 and six children of unknown status. The arrow indicates the patient described in this section (proband). (B) CT of the younger sibling taken at age 12. Extensive diffuse ground-glass opacities with subpleural sparing and enlargement of the main pulmonary artery are observed. (C) Schematic localization of the ISG15 variant in the genomic DNA locus indicated by the arrow. (D) Expression levels of various ISGs ( IFI27 , MX1 , SIGLEC1 , and IFIT1 ) were assessed from patient’s whole blood, as well as from the three healthy controls. Target genes were normalized on 18S rRNA expression. (E) 3′-RACE PCR targeting ISG15 was performed on RNA isolated from patient’s whole blood. (F) Sanger sequencing of the 3′-RACE band from E. (G) HEK293T cells were transfected with a plasmid encoding the ISG15 variant (c.463insC), ISG15 WT, or Luc. Relative mRNA levels for ISG15 were assessed by qRT-PCR, performed twice for each variant, with technical triplicates; the data from one representative experiment ( n = 3) are shown. The expression level of ISG15 was normalized to GAPDH . (H) HEK293T cells were transfected with a plasmid encoding the ISG15 variant (c.463insC), ISG15 WT or Luc, plus HERC5, UBE1L, and UBCH8, as described in . Cell lysates were analyzed by western blotting for ISG15 and ISGylation; a representative experiment is shown. CT, chest tomography. Source data are available for this figure: .

Article Snippet: The probes used were the following: TGFB1 Hs07289533_m1, ISG15 Hs01921425_s1, IFIT1 Hs03027069_s1, IFI27 Hs01086373_g1, SIGLEC1 Hs00988063_m1, MX1 Hs00895608_m1, FGF-2 Hs00266645_m1, ITGB6 Hs00168458_m1, PDGFA Hs00234994_m1.

Techniques: Mutagenesis, Variant Assay, Expressing, Isolation, Sequencing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Tomography

Journal: Journal of Human Immunity

Article Title: Human ISG15 deficiency unveils impaired healing of ulcerations via type I interferon–mediated fibrosis

doi: 10.70962/jhi.20250011

Figure Lengend Snippet: Myofibroblast activation in ISG15-deficient cells. (A) Spatial expression and intensity levels of two myofibroblast markers ( ACTA2 and COL5A1 ). (B) Relative expression of various myofibroblast markers for clusters 1 and 5 separated by sample biopsy. (C) Percentage of total spots that express TGFβ in the entire biopsy, within CD68 + spots only, and within myofibroblast cluster 5. (D) Significance of pathway enrichment for clusters 0, 1, 5, 3, 7, and 9 generated by MSigDB Hallmark 2020 and BioPlanet 2019 analysis from Enrichr. Adjusted P values are shown. (E) Relative RNA levels of myofibroblast markers ( PDGFA , ITGB6 , FGF2 ) following a 72-h treatment with 1,000 IU/ml IFNα2b, 10 ng/ml TGFβ, or both of WT and ISG15 KO lung epithelial cells (A549). (F) Percentage of cells undergoing EMT upon TGFβ/IFNα stimulation. (G) Relative RNA levels of myofibroblast markers ( PDGFA , ITGB6 , FGF2 ) following a 5-day treatment with 1,000 IU/ml IFNα2b, 10 ng/ml TGFβ, or both of WT and ISG15 KO fibroblasts. (H) ACTA2 (myofibroblasts) IHC for lesion 1 and lesion 3. P values were calculated with two-tailed t test. *P < 0.05.

Article Snippet: The probes used were the following: TGFB1 Hs07289533_m1, ISG15 Hs01921425_s1, IFIT1 Hs03027069_s1, IFI27 Hs01086373_g1, SIGLEC1 Hs00988063_m1, MX1 Hs00895608_m1, FGF-2 Hs00266645_m1, ITGB6 Hs00168458_m1, PDGFA Hs00234994_m1.

Techniques: Activation Assay, Expressing, Generated, Two Tailed Test

Journal: Journal of Human Immunity

Article Title: Human ISG15 deficiency unveils impaired healing of ulcerations via type I interferon–mediated fibrosis

doi: 10.70962/jhi.20250011

Figure Lengend Snippet: IFNα induces apoptosis in ISG15 KO cells. (A) Healing/migration ability of WT and ISG15 KO fibroblasts was assessed with the scratch assay. Left; percent confluency was recorded every hour for a total of 93 h. Right; experimental replicates at 48, 72, and 93 h after scratch. (B) Representative images of WT and ISG15 KO fibroblasts at 1 h (left), 49 h (middle), and 92 h (right) of 1,000 IU ml −1 IFNα treatment. White arrows indicate macroscopic evidence of cellular stress. (C) WT and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h, and cell death was quantified by measuring Annexin V and Propidium Iodide (PI) levels through flow cytometry. (D) Control and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h following a 30-min pretreatment with necrostatin-1, and cell death was quantified by measuring Annexin V and PI levels through flow cytometry. (E) Metabolic activity of two control and one ISG15 KO fibroblast cell lines was assessed with the Deep Blue assay. Nec-1; necrostatin-1. (F) Control and ISG15-deficient fibroblasts were treated with either IFNα2b (1, 100, 1,000 IU/ml) or chloroquine (0, 25, 50, 100 μM) for 72 h. Cell lysates were analyzed by western blotting for autophagy (LC3B). (G) Control and ISG15-deficient fibroblasts were treated with either IFNα2b (100 IU/ml), or TNFα (5 ng/μl), or both for 72 h, and percent death was assessed with the Deep Blue assay as compared to the nontreated condition for each variant. (H) WT and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h, and cell death was quantified by measuring cleaved (active) caspase 3 levels through flow cytometry. Source data are available for this figure: . P values were calculated with two-tailed t test. **P < 0.01; ***P < 0.001.

Article Snippet: The probes used were the following: TGFB1 Hs07289533_m1, ISG15 Hs01921425_s1, IFIT1 Hs03027069_s1, IFI27 Hs01086373_g1, SIGLEC1 Hs00988063_m1, MX1 Hs00895608_m1, FGF-2 Hs00266645_m1, ITGB6 Hs00168458_m1, PDGFA Hs00234994_m1.

Techniques: Migration, Wound Healing Assay, Flow Cytometry, Control, Activity Assay, Western Blot, Variant Assay, Two Tailed Test

Journal: Journal of Human Immunity

Article Title: Human ISG15 deficiency unveils impaired healing of ulcerations via type I interferon–mediated fibrosis

doi: 10.70962/jhi.20250011

Figure Lengend Snippet: Reconstitution with WT ISG15 and inhibition of apoptosis rescue apoptosis of IFNα-treated ISG15 KO cells. (A) Healing/migration ability of WT, UBE1L KO, and ISG15 KO fibroblasts, as well as ISG15 KO cells complemented with WT ISG15 or Luc, was assessed with the scratch assay for 93 h. (B) Apoptosis following 72-h IFNα2b treatment of WT, UBE1L KO, and ISG15 KO fibroblasts, as well as ISG15 KO cells complemented with WT ISG15 or Luc, was assessed by quantifying cleaved caspase 3 levels through flow cytometry. (C) Rescue of A549 ISG15 KO IFNα2b-induced cell death with pan-caspase inhibitor (Z-VAD). (D) Proliferation of ISG15 KO lung epithelial cells (A549) with or without IFNα2b as compared to control. (E) CASP3 IHC for three healthy volunteer and three patient’s skin biopsies. White arrows indicate areas of cleaved Caspase-3 (brown).

Article Snippet: The probes used were the following: TGFB1 Hs07289533_m1, ISG15 Hs01921425_s1, IFIT1 Hs03027069_s1, IFI27 Hs01086373_g1, SIGLEC1 Hs00988063_m1, MX1 Hs00895608_m1, FGF-2 Hs00266645_m1, ITGB6 Hs00168458_m1, PDGFA Hs00234994_m1.

Techniques: Inhibition, Migration, Wound Healing Assay, Flow Cytometry, Control

Journal: bioRxiv

Article Title: ENPP1 buffers extracellular cGAMP in brown adipose tissue to limit insulin resistance

doi: 10.64898/2026.04.12.718013

Figure Lengend Snippet: a , Schematic of brown adipocyte differentiation (left), and relative Ucp1 expression as a marker of brown adipocytes in primary brown preadipocytes and differentiated brown adipocytes, normalized to levels in differentiated brown adipocytes (right). n = 3 independent experiments. Data are presented as mean ± s.d. b, Western blot of WCE and cytosolic fractions from brown preadipocytes and differentiated brown adipocytes using cytosolic marker GAPDH, mitochondrial marker TOM20 and nuclear marker H3 (left). Relative cytosolic mtDNA markers (Cytb, D-loop, mt-Tf, ND4) normalized to H19 in WCE, then normalized to levels of brown preadipocytes (right). n = 2 independent experiments, 3 biological replicates each. Data are presented as mean ± s.d. c, Schematic of in vitro cGAMP measurement (left). Intracellular cGAMP in lysates of brown preadipocytes and differentiated brown adipocytes cultured with or without ENPP1 inhibitor (1 µM STF-1623) over 2 days (middle). Extracellular cGAMP in supernatants of brown preadipocytes and differentiated brown adipocytes cultured with or without ENPP1 inhibitor (1 µM STF-1623) over 2 days (right). bdl, 9 pM. n = 3 (intracellular) or 4 (extracellular) independent experiments. Data are presented as mean ± s.d. d, Relative Ifit1 , Cxcl10 , and Isg15 expression as markers of cGAMP-STING pathway activation in primary brown preadipocytes and differentiated brown adipocytes, normalized to levels in differentiated brown adipocytes. n = 3 independent experiments. Data are presented as mean ± s.d. e, Schematic model of brown adipocytes as a source of extracellular cGAMP driven by cytosolic mtDNA. Statistical significance was assessed using two-sided unpaired t tests. WCE, whole cell extract; cyto, cytosolic.

Article Snippet: Probes from Thermo Fisher include the following: Ucp1 Mm01244861_m1), Isg15 (Mm01705338_s1), Cxcl10 (Mm00445235_m1), Ifit1 (Mm07295796_m1).

Techniques: Expressing, Marker, Western Blot, In Vitro, Cell Culture, Activation Assay

Journal: bioRxiv

Article Title: STING causes replication stress and nascent DNA degradation via SAMHD1

doi: 10.64898/2026.03.28.714577

Figure Lengend Snippet: ( A ) Western blot analysis of subcellular fractions from HDF treated for 6 days with doxycycline (Dox) to induce progerin expression, for 24 h with hydroxyurea (HU), or transfected with single-stranded DNA (ssDNA). Fraction markers include β-tubulin (cytoplasm), SEC61 (membrane), Lamin A (nucleus), and Histone H3 (chromatin). Note the increased presence of STING at nucleus and chromatin upon replication stress. Membranes imaged with prolonged exposure (high exposure) show a marked increase in signal intensity. ( B ) Immunofluorescence (IF) with STING antibody in HDF treated with vehicle, ssDNA, HU, Doxy (progerin), or dsDNA. ( C ) Quantification of cGAMP levels measured by ELISA in HDF treated as indicated. ( D ) IF with STING antibody and quantification of percentage of cells showing STING localization to the perinuclear compartment (PNC) upon different treatments. ( E ) IF with antibody recognizing phosphorylated STING on Ser366 and quantification of percentage of cells positive for S366 p-STING. ( F ) Immunoblot analysis of STING, GFP-progerin, and markers of activation of the canonical cGAS-STING pathway ( S366 p-STING, S386 p-IRF3, and S172 p-TBK1) following treatments. ( G ) Immunoblot analysis of STING pathway components and ISG proteins (STAT1, S727 p-STAT1, RIG-I, and ISG15) after indicated treatments.

Article Snippet: Primary antibodies used were β-tubulin (1:5000- Origene-AP31823PU-N), GAPDH (1:1000- Cell Signaling-2118), Lamina A (1:3000- Abcam-1791), Progerin (1:1000 Santa Cruz-81511), ISG15 (1:1000 Santa Cruz-166755), STING (1:1000-Cell Signaling-13647), S366 p-STING (1:1000- Cell Signaling- 50907), RIG-I (1:1000-Cell Signaling-3743S), S33-p-RPA (1:1000 Bethyl- PLA0070), SAMHD1 (1:1000- Cell Singaling-49158), λH2AX (1:1000- Cell Signaling- 2577).

Techniques: Western Blot, Expressing, Transfection, Membrane, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: bioRxiv

Article Title: STING causes replication stress and nascent DNA degradation via SAMHD1

doi: 10.64898/2026.03.28.714577

Figure Lengend Snippet: ( A ) U2OS tumor cells were generated that express progerin, STING, or both, using empty vectors (EV) as controls. Immunoblots performed to assess replication stress marker 33 p-RPA, and activation of an IFN response (ISG15, RIG-I). ( B ) DNA fiber assays to monitor total tract length in U2OS +/- progerin and +/- STING. ( C ) Fork symmetry (ratio of the sisters CldU tracts from bidirectional forks) in same conditions as in (B). ( D ) DNA fiber analysis to monitor total tract length in U2OS +/- progerin and +/- STING with or without dNTP supplementation. ( E ) Fork symmetry in the same samples as in (D). ( F ) Immunoblot showing STING expression and SAMHD1 knockdown in control and progerin-expressing U2OS cells. ( G ) DNA fiber analysis to monitor total tract length in U2OS +/- progerin and +/- STING with or without SAMHD1 depletion. ( H ) Fork symmetry in the same samples as in (G). ( I ) Subcellular fractionation of U2OS +/- progerin and +/- STING, probed for STING and SAMHD1. H3 and SEC61 were used as nuclear and ER markers, respectively. For all quantifications, each point represents a single replication fork; n = 3 biologically independent experiments.

Article Snippet: Primary antibodies used were β-tubulin (1:5000- Origene-AP31823PU-N), GAPDH (1:1000- Cell Signaling-2118), Lamina A (1:3000- Abcam-1791), Progerin (1:1000 Santa Cruz-81511), ISG15 (1:1000 Santa Cruz-166755), STING (1:1000-Cell Signaling-13647), S366 p-STING (1:1000- Cell Signaling- 50907), RIG-I (1:1000-Cell Signaling-3743S), S33-p-RPA (1:1000 Bethyl- PLA0070), SAMHD1 (1:1000- Cell Singaling-49158), λH2AX (1:1000- Cell Signaling- 2577).

Techniques: Generated, Western Blot, Marker, Activation Assay, Expressing, Knockdown, Control, Fractionation

Journal: bioRxiv

Article Title: STING causes replication stress and nascent DNA degradation via SAMHD1

doi: 10.64898/2026.03.28.714577

Figure Lengend Snippet: ( A ) Western blot analysis of subcellular fractions from HDF treated for 6 days with doxycycline (Dox) to induce progerin expression, for 24 h with hydroxyurea (HU), or transfected with single-stranded DNA (ssDNA). Fraction markers include β-tubulin (cytoplasm), SEC61 (membrane), Lamin A (nucleus), and Histone H3 (chromatin). Note the increased presence of STING at nucleus and chromatin upon replication stress. Membranes imaged with prolonged exposure (high exposure) show a marked increase in signal intensity. ( B ) Immunofluorescence (IF) with STING antibody in HDF treated with vehicle, ssDNA, HU, Doxy (progerin), or dsDNA. ( C ) Quantification of cGAMP levels measured by ELISA in HDF treated as indicated. ( D ) IF with STING antibody and quantification of percentage of cells showing STING localization to the perinuclear compartment (PNC) upon different treatments. ( E ) IF with antibody recognizing phosphorylated STING on Ser366 and quantification of percentage of cells positive for S366 p-STING. ( F ) Immunoblot analysis of STING, GFP-progerin, and markers of activation of the canonical cGAS-STING pathway ( S366 p-STING, S386 p-IRF3, and S172 p-TBK1) following treatments. ( G ) Immunoblot analysis of STING pathway components and ISG proteins (STAT1, S727 p-STAT1, RIG-I, and ISG15) after indicated treatments.

Article Snippet: Primary antibodies used were β-tubulin (1:5000- Origene-AP31823PU-N), GAPDH (1:1000- Cell Signaling-2118), Lamina A (1:3000- Abcam-1791), Progerin (1:1000 Santa Cruz-81511), ISG15 (1:1000 Santa Cruz-166755), STING (1:1000-Cell Signaling-13647), S366 p-STING (1:1000- Cell Signaling- 50907), RIG-I (1:1000-Cell Signaling-3743S), S33-p-RPA (1:1000 Bethyl- PLA0070), SAMHD1 (1:1000- Cell Singaling-49158), λH2AX (1:1000- Cell Signaling- 2577).

Techniques: Western Blot, Expressing, Transfection, Membrane, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: bioRxiv

Article Title: STING causes replication stress and nascent DNA degradation via SAMHD1

doi: 10.64898/2026.03.28.714577

Figure Lengend Snippet: ( A ) U2OS tumor cells were generated that express progerin, STING, or both, using empty vectors (EV) as controls. Immunoblots performed to assess replication stress marker 33 p-RPA, and activation of an IFN response (ISG15, RIG-I). ( B ) DNA fiber assays to monitor total tract length in U2OS +/- progerin and +/- STING. ( C ) Fork symmetry (ratio of the sisters CldU tracts from bidirectional forks) in same conditions as in (B). ( D ) DNA fiber analysis to monitor total tract length in U2OS +/- progerin and +/- STING with or without dNTP supplementation. ( E ) Fork symmetry in the same samples as in (D). ( F ) Immunoblot showing STING expression and SAMHD1 knockdown in control and progerin-expressing U2OS cells. ( G ) DNA fiber analysis to monitor total tract length in U2OS +/- progerin and +/- STING with or without SAMHD1 depletion. ( H ) Fork symmetry in the same samples as in (G). ( I ) Subcellular fractionation of U2OS +/- progerin and +/- STING, probed for STING and SAMHD1. H3 and SEC61 were used as nuclear and ER markers, respectively. For all quantifications, each point represents a single replication fork; n = 3 biologically independent experiments.

Article Snippet: Primary antibodies used were β-tubulin (1:5000- Origene-AP31823PU-N), GAPDH (1:1000- Cell Signaling-2118), Lamina A (1:3000- Abcam-1791), Progerin (1:1000 Santa Cruz-81511), ISG15 (1:1000 Santa Cruz-166755), STING (1:1000-Cell Signaling-13647), S366 p-STING (1:1000- Cell Signaling- 50907), RIG-I (1:1000-Cell Signaling-3743S), S33-p-RPA (1:1000 Bethyl- PLA0070), SAMHD1 (1:1000- Cell Singaling-49158), λH2AX (1:1000- Cell Signaling- 2577).

Techniques: Generated, Western Blot, Marker, Activation Assay, Expressing, Knockdown, Control, Fractionation