Journal: The Journal of investigative dermatology

Article Title: Delineating the Healthy Human Skin UV Response and Early Induction of Interferon Pathway in Cutaneous Lupus Erythematosus.

doi: 10.1016/j.jid.2019.02.035

Figure Lengend Snippet: Figure 2. Altered UV photoprovocation-response genes in DLE and SCLE. (a) The upregulated genes (yellow box) in the known SLE pathway [KEGG: hsa05322]. (b) Ten most enriched gene sets in the LINCS L1000 ligand perturbation dataset and the altered genes corresponding to the enriched gene sets; each gene set consists of differentially regulated genes induced by a ligand in a cell line. (c) Upregulation of IFIT1, IFIT2, and IFIT3 mRNA expression in photoprovocated SCLE (in all but two) and DLE skin lesions by qRT-PCR validation. (d) IFIT protein expression in skin samples by immunohistochemistry. IFIT proteins are more strongly expressed in SCLE than in healthy controls after UV provocation. Note the strong expression (dark brown) especially in basal keratinocytes and in areas with apoptotic keratinocytes (i.e., cells with pyknotic nuclei and eosinophilic cytoplasm). Scale bar ¼ 100 mm. DLE, discoid lupus erythematosus; qRT-PCR, quantitative real-time reverse transcriptaseePCR; SCLE, subacute cutaneous lupus erythematosus; SLE, systemic lupus erythematosus.

Article Snippet: Primary polyclonal rabbit antibodies to IFIT1, IFIT2, and IFIT3 (Sigma/Atlas antibodies, St. Louis, MO) were diluted in 1% BSA and applied on the sections overnight at 4 C. The bound antibodies were visualized using Vector Universal ImmPress Kit and Vector NovaRed (Vector Laboratories, Burlingame, CA), and counterstained with hematoxylineeosin.

Techniques: Expressing, Quantitative RT-PCR, Biomarker Discovery, Immunohistochemistry

Journal: Molecular Neurodegeneration

Article Title: Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

doi: 10.1186/s13024-018-0263-7

Figure Lengend Snippet: Endogenous dsRNA response and type I interferon promote paraspeckle hyper-assembly in stable cell lines. a and b Depletion of TDP-43, Dicer, Drosha, ADAR1 but not Ago2 or FUS causes intracellular build-up of dsRNA. dsRNA was detected by immunocytochemistry using J2 antibody. Representative images of all conditions are shown. Scale bars, 50 and 10 μm for general plane and close-up panels respectively. c Levels of Alu-containing RNA as analysed by qRT-PCR using specific primers recognising Alu elements (n = 4). *p < 0.05 (Mann-Whitney U -test). d and e Markers of activated cellular reponse to dsRNA are upregulated in TDP-43 depleted cells. Levels of phosphorylated PKR and eIF2α were analysed by Western blot ( d , representative blots are shown) and expression of IFNB1 and an IFN-stimulated gene CXCL10 - by qRT-PCR ( e , n = 6). *p < 0.05 (Mann-Whitney U -test). f IFNbeta treatment stimulates NEAT1 expression and paraspeckle formation. NEAT1 levels were measured by qRT-PCR (n = 6). **p < 0.01 (Mann-Whitney U -test). Staining for an IFN-inducible protein IFIT3 was used as a positive control. Scale bar, 10 μm. g Simultaneous IFNbeta knockdown partially reverses the effect of TDP-43 depletion on paraspeckles. * and #p < 0.05, ***p < 0.001 (one-way ANOVA with Holm-Sidak correction for multiple comparisons). Scale bar, 10 μm. In all panels, cells were harvested for analysis 48 h post-transfection. Paraspeckles in panels f and g were visualised by NEAT1_2 RNA-FISH

Article Snippet: The following commercial primary antibodies were used: TDP-43 (rabbit polyclonal, 10782–2-AP, Proteintech and mouse monoclonal, MAB7778-SP, R&D Biosystems); FUS (rabbit polyclonal, Proteintech, 11570–1-AP); p54nrb/NONO (rabbit polyclonal C-terminal, Sigma); PSF/SFPQ (rabbit monoclonal, ab177149, Abcam); Tuj (β-Tubulin III, mouse monoclonal, Sigma); dsRNA (mouse monoclonal, J2, Kerafast); cleaved caspase 3 (rabbit polyclonal, 9661, Cell Signaling); NF-κB p65 (rabbit monoclonal, D14E12, Cell Signaling); IFIT3 (rabbit polyclonal, Bethyl); p-eIF2α (rabbit monoclonal, ab32157, Abcam); p-PKR (rabbit polyclonal, Thr451, ThermoFisher); PKR (mouse monoclonal, MAB1980-SP, R&D Systems); eIF2α (rabbit monoclonal, D7D3, Cell Signaling); β-actin (mouse monoclonal, A5441, Sigma).

Techniques: Stable Transfection, Immunocytochemistry, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing, Staining, Positive Control, Knockdown, Transfection

Journal: Journal of Neuroinflammation

Article Title: Progression of pathology in PINK1-deficient mouse brain from splicing via ubiquitination, ER stress, and mitophagy changes to neuroinflammation

doi: 10.1186/s12974-017-0928-0

Figure Lengend Snippet: Systematic analysis of key factors in the mitochondria-associated innate immunity pathway, using acute starvation of human SH-SY5Y neuroblastoma cells to reveal the transcriptional regulation of stress responses and their dependence on PINK1. Transcript levels were documented in control non-target knock-down (NT-KD) versus PINK1 -knock-down ( PINK1 -KD) cells for the following inflammatory factors. a Summary scheme on the detection of pathogen DNA/RNA in the cytosol and the mitochondria-associated triggering of innate immunity, as altered during starvation by PINK1. Viral RNA (ssRNA or dsRNA) is recognized in the cytosol by helicase RIG-I and MDA5. This triggers the dimerization of MAVS in the outer mitochondrial membrane (OMM) which recruits transactivators (such as TRADD, TRAF, IKK family) leading to nuclear translocation of phosphorylated IRF3, IRF7, and NF-κB and promoting the transcription of IFN stimulated genes and pro-inflammatory cytokines, respectively. Effector molecules such as IFIT1, IFIT2, IFIT3, and RSAD2 (viperin) inhibit virus DNA/RNA replication. IFIT3 also functions as a scaffold to facilitate interaction between MAVS and TBK1 and represents a positive feedback of DDX58 (RIG-I) signaling through MAVS. Low ΔΨm or decreased ROS inhibit MAVS-mediated signaling. Mitochondria cooperate with the endoplasmic reticulum (ER) to regulate lipid synthesis and antiviral signaling at the mitochondria-associated membranes (MAM), possibly by interactions of MAVS/MFN1 with TMEM173 (STING). MFN1 leads to the redistribution of MAVS along mitochondria and a fusion of the mitochondrial network that promotes the interaction between MAVS and STING. Low energy induces the localization of PINK1 to the OMM and recruitment of PARKIN from the cytosol, which is the signal for dysfunctional mitochondria to be digested in the autophagosome. Abbreviations: 5′ ppp 5′ triphosphate, ΔΨm mitochondrial membrane potential, IκB inhibitor of κ light polypeptide gene enhancer in B cells, IKK IκB Kinase, LC3II phosphatidylethanolamine conjugate of the autophagy-related protein LC3 (MAP1LC3), MDA5 melanoma differentiation-associated gene 5, NF-κB nuclear factor κB, p62 sequestosome-1, adaptor between polyubiquitinated substrates and autophagic machinery, PARKIN ubiquitin ligase, its loss-of-function leads to the PARK2 variant of Parkinson’s disease, RIG-1 (DDX58) retinoic acid-inducible gene-1 (DExD/H-Box RNA Helicase 58), ROS reactive oxygen species, ssRNA/dsRNA single-stranded/double-stranded RNA, TMEM173 (STING) transmembrane Protein 173, TRADD tumor necrosis factor receptor type 1 associated death domain protein, TRAF TNF-receptor-associated factor . b PINK1 (PTEN induced kinase 1) as a known determinant of selective mitophagy and autosomal recessive Parkinson’s disease; HPRT1 (Hypoxanthine Phosphoribosyltransferase 1) as a loading control. c RSAD2 (= viperin, radical S-adenosyl methionine domain containing 2) as an interferon-inducible lipid-droplet associated virus inhibitory factor. d HEBP1 (heme binding protein 1) that contains a natural ligand for formyl peptide receptor-like receptor 2. e TBK1 (TANK-binding kinase 1) that phosphorylates interferon regulatory factors in response to toll-like receptor activation. f IRF3 (interferon regulatory factor 3) as a regulator of type I Interferon gene transcription. g MFN1 (mitofusin 1) as PARKIN-dependent factor in mitochondrial dynamics and mitochondria-associated anti-microbial signaling. h IFIT3 (interferon-induced protein with tetratricopeptide repeats 3) as a detector of pathogen DNA/RNA. i IFIT1 (interferon-induced protein with tetratricopeptide repeats 3) as a detector of pathogen DNA/RNA. j MAVS (mitochondria-associated viral sensor) as inducer of interferon-dependent long-term expression of defense factors. k – m The levels of known autophagy factors during this starvation time course exhibited progressive consumption of p62 in spite of its transcriptional induction, in parallel to a PINK1 -modulated transcriptional induction of LRRK2. Four independent experiments compared their expression during a nutrient and trophic deprivation time course triggered by a culture switch from RPMI growth medium to HBSS starvation medium. The bar graphs show mean and standard error of the mean, illustrating the significance with asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: RNA was isolated with the RNeasy mini kit (Qiagen) and then treated with DNase I. cDNA was synthesized with SuperScript III reverse transcriptase using oligo(dT) 20 and random primers (Invitrogen). cDNA from 20 to 25 ng RNA were utilized in a 20 μl reaction volume using the StepOnePlus Real-Time PCR System and the appropriate murine (lowercase) or human (uppercase) TaqMan gene expression assays (Applied Biosystems): for mouse Pink1 (Mm00550827_m1), Creb3 (Mm00501607_m1), Ddx58 (Mm01216853_m1), Hebp1 (Mm00469161_m1), Ifit1 (Mm00515153_m1), Ifit3 (Mm01704846_s1), Irf3 (Mm00516784_m1), Mapk8 (Mm01218957_m1, Mm01218946_m1, Mm00489514_m1), Mapk9 (Mm00444239_m1), Mapk14 (Mm01301009_m1), Mavs (Mm00523170_m1), Mfn1 (Mm00612599_m1), Nfkbia (Mm00477798_m1), Rsad2 (Mm00491265_m1), Srsf10 (Mm01193320_m1), Tbk1 (Mm00451150_m1), Tnf (Mm00443258_m1), for human PINK1 (Hs00260868_m1), DDX58 (Hs01061436_m1), HEBP1 (Hs00211123_m1), IFIT1 (Hs03027069_s1), IFIT3 (Hs00155468_m1), IRF3 (Hs01547283_m1), LRRK2 (Hs00411197_m1), MAVS (Hs00920075_m1), MFN1 (Hs00966851_m1), RSAD2 (Hs00369813_m1), SQSTM1 (Hs00177654_m1), TBK1 (Hs00179410_m1). mRNA expression was normalized to the TATA binding protein gene expression or the Hypoxanthine Phosphoribosyltransferase 1 gene expression ( Tbp : Mm00446973_m1, TBP : Hs99999910_m1, HPRT1: Hs99999909_m1).

Techniques: Control, Knockdown, Membrane, Translocation Assay, Virus, Ubiquitin Proteomics, Variant Assay, Binding Assay, Activation Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: Progression of pathology in PINK1-deficient mouse brain from splicing via ubiquitination, ER stress, and mitophagy changes to neuroinflammation

doi: 10.1186/s12974-017-0928-0

Figure Lengend Snippet: Poly(I:C) triggered expression responses of innate immunity factors in dependence on PINK1. a Transcriptional changes: SH-SY5Y human neuroblastoma (above) and murine embryonal fibroblast cells (below) were studied regarding the expression of key inflammatory factors in untreated versus drug-treated cells, comparing control with PINK1-deficiency ( n = 8 each for RSAD2 , DDX58 , IFIT3 , and IFIT1 in man, and n = 4 each for Rsad2 , Ddx58 , Ifit3 , Ifit1 in mouse). TBP transcript levels were used as loading controls to normalize the data. The bar graphs show mean and standard error of the mean, illustrating the significance with asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). b Protein changes: representative quantitative immunoblots and their statistical evaluation in bar graphs ( n = 3 WT versus 4 mutant) showed a massive induction of the RNA sensors RSAD2, DDX58, IFIT3 (in human as well as mouse), and IFIT1 (human-specific antibody), which was blunted in PINK1-deficient cells (significance demonstrable only in neural cells). The beta-Actin protein level was used as loading control to normalize the quantitative results

Article Snippet: RNA was isolated with the RNeasy mini kit (Qiagen) and then treated with DNase I. cDNA was synthesized with SuperScript III reverse transcriptase using oligo(dT) 20 and random primers (Invitrogen). cDNA from 20 to 25 ng RNA were utilized in a 20 μl reaction volume using the StepOnePlus Real-Time PCR System and the appropriate murine (lowercase) or human (uppercase) TaqMan gene expression assays (Applied Biosystems): for mouse Pink1 (Mm00550827_m1), Creb3 (Mm00501607_m1), Ddx58 (Mm01216853_m1), Hebp1 (Mm00469161_m1), Ifit1 (Mm00515153_m1), Ifit3 (Mm01704846_s1), Irf3 (Mm00516784_m1), Mapk8 (Mm01218957_m1, Mm01218946_m1, Mm00489514_m1), Mapk9 (Mm00444239_m1), Mapk14 (Mm01301009_m1), Mavs (Mm00523170_m1), Mfn1 (Mm00612599_m1), Nfkbia (Mm00477798_m1), Rsad2 (Mm00491265_m1), Srsf10 (Mm01193320_m1), Tbk1 (Mm00451150_m1), Tnf (Mm00443258_m1), for human PINK1 (Hs00260868_m1), DDX58 (Hs01061436_m1), HEBP1 (Hs00211123_m1), IFIT1 (Hs03027069_s1), IFIT3 (Hs00155468_m1), IRF3 (Hs01547283_m1), LRRK2 (Hs00411197_m1), MAVS (Hs00920075_m1), MFN1 (Hs00966851_m1), RSAD2 (Hs00369813_m1), SQSTM1 (Hs00177654_m1), TBK1 (Hs00179410_m1). mRNA expression was normalized to the TATA binding protein gene expression or the Hypoxanthine Phosphoribosyltransferase 1 gene expression ( Tbp : Mm00446973_m1, TBP : Hs99999910_m1, HPRT1: Hs99999909_m1).

Techniques: Expressing, Control, Western Blot, Mutagenesis

Journal: Journal of Neuroinflammation

Article Title: Progression of pathology in PINK1-deficient mouse brain from splicing via ubiquitination, ER stress, and mitophagy changes to neuroinflammation

doi: 10.1186/s12974-017-0928-0

Figure Lengend Snippet: Altered antiviral factors in primary skin fibroblasts from PARK6 patients. a The levels of RSAD2 , DDX58 , and IFIT3 transcript as well as ( b ) the levels of RSAD2, DDX58, and IFIT3 protein were assessed in unstressed cells from 3 homozygous G309D-PINK1 PD patients versus matched controls. The bar graphs show mean and standard error of the mean, illustrating the significance with asterisks (* p < 0.05 and ** p < 0.01)

Article Snippet: RNA was isolated with the RNeasy mini kit (Qiagen) and then treated with DNase I. cDNA was synthesized with SuperScript III reverse transcriptase using oligo(dT) 20 and random primers (Invitrogen). cDNA from 20 to 25 ng RNA were utilized in a 20 μl reaction volume using the StepOnePlus Real-Time PCR System and the appropriate murine (lowercase) or human (uppercase) TaqMan gene expression assays (Applied Biosystems): for mouse Pink1 (Mm00550827_m1), Creb3 (Mm00501607_m1), Ddx58 (Mm01216853_m1), Hebp1 (Mm00469161_m1), Ifit1 (Mm00515153_m1), Ifit3 (Mm01704846_s1), Irf3 (Mm00516784_m1), Mapk8 (Mm01218957_m1, Mm01218946_m1, Mm00489514_m1), Mapk9 (Mm00444239_m1), Mapk14 (Mm01301009_m1), Mavs (Mm00523170_m1), Mfn1 (Mm00612599_m1), Nfkbia (Mm00477798_m1), Rsad2 (Mm00491265_m1), Srsf10 (Mm01193320_m1), Tbk1 (Mm00451150_m1), Tnf (Mm00443258_m1), for human PINK1 (Hs00260868_m1), DDX58 (Hs01061436_m1), HEBP1 (Hs00211123_m1), IFIT1 (Hs03027069_s1), IFIT3 (Hs00155468_m1), IRF3 (Hs01547283_m1), LRRK2 (Hs00411197_m1), MAVS (Hs00920075_m1), MFN1 (Hs00966851_m1), RSAD2 (Hs00369813_m1), SQSTM1 (Hs00177654_m1), TBK1 (Hs00179410_m1). mRNA expression was normalized to the TATA binding protein gene expression or the Hypoxanthine Phosphoribosyltransferase 1 gene expression ( Tbp : Mm00446973_m1, TBP : Hs99999910_m1, HPRT1: Hs99999909_m1).

Techniques:

Journal: Scientific Reports

Article Title: Interferon-induced protein with tetratricopeptide repeats 3 may be a key factor in primary biliary cholangitis

doi: 10.1038/s41598-021-91016-6

Figure Lengend Snippet: IFIT3 knockdown increased cell proliferation, apoptosis and decreased in cellular senescence in BECs treated with serum depletion and GCDC. ( A , B ) Effective knockdown of Ifit3. ( A ) The expression of Ifit3 mRNA in BECs treated with serum depletion and Ifit3 small interfering RNA ( si RNA) or control si RNA for 1 day. Ifit3 mRNA was significantly increased in BECs treated with serum depletion (Dep) compared to the control ( p < 0.01) and the increase was significantly suppressed by a treatment with Ifit3 si RNA compared to the control siRNA ( p < 0.01). Data are expressed as the means ± SD. * p < 0.01 vs. control + Cont si RNA, # p < 0.01 compared to Dep + Ifit3 si RNA. n = 3 for each group. ( B ) The protein level expression of IFIT3 assessed by immunoblotting in BECs treated with serum depletion and Ifit3 si RNA or control si RNA for 4 days. The protein level expression of IFIT3 was significantly increased in BECs treated with serum depletion compared to the control ( p < 0.05) and the increase was significantly suppressed by a treatment with Ifit3 si RNA compared to the control siRNA ( p < 0.05). * p < 0.05 vs. control + Cont si RNA; # p < 0.05 compared to Dep + Ifit3 si RNA, n = 3 for each group. ( C ) Cell proliferation was increased by a treatment with Ifit3 si RNA. Cell proliferation activity was detected by BrdU assay. Cell proliferation activities of BECs after the induction of cellular senescence (serum depletion and GCDC) for 4 days with a treatment with Ifit3 si RNA or control si RNA. BrdU-LI is significantly lower in BECs treated with serum depletion or GCDC compared to the control ( p < 0.01) and significantly higher in BECs treated with Ifit3 si RNA. The data are expressed as the mean ± SD. * p < 0.05 vs. Cont si RNA; # p < 0.05. n = 5 for each group. (Con-Con si , 7.75 ± 2.95; Con-Ifit3 si , 7.75 ± 2.50; Dep-Cont si , 0.72 ± 1.10; Dep-Ifit3 si , 3.71 ± 1.84; GCDC-Cont si , 1.53 ± 1.94; Dep-Ifit3 si , 7.60 ± 3.42). ( D ) Cell growth curve assessed by cell number was not changed by a treatment with Ifit3 si RNA. Cell number was assessed by WST assay after the induction of cellular senescence with serum deprivation (Dep) or GCDC (500 nM) with Ifit3 si RNA or control si RNA for 1, 2, 4 and 7 days. The data are expressed as the mean ± SD. * p < 0.01 vs. Con-Cont si RNA. n = 4 for each group. ( E ) G1/S arrest was resolved by a treatment with IFIT3 si RNA. Cell cycle was analyzed using Cell-Clock cell cycle assay on BECs by the treatment with serum depletion (Dep) or GCDC for 4 days with or without knockdown of IFIT3 using si RNA. Cells become yellow in G1, green in S/G2 and blue in M phase. The data are expressed as the mean ± SD. * p < 0.01 vs. Con-Con si RNA; # p < 0.05 between IFIT3 siRNA and Con si RNA in each condition. n = 4 for each group. (% S-phase: Con-Con si , 27.4 ± 2.2; Con-Ifit3 si , 22.9 ± 4.0; Dep-Cont si , 8.6 ± 3.3; Dep-Ifit3 si , 25.3 ± 1.4; GCDC-Cont si , 11.7 ± 3.3; Dep-Ifit3 si , 26.6 ± 3.3). ( F ) Apoptosis was effectively induced in senescent BECs by a treatment with Ifit3 si RNA. Apoptosis was assessed by detecting caspase-3/7 activity after the induction of cellular senescence with serum deprivation (Dep) or GCDC (500 nM) with Ifit3 si RNA or control si RNA for 4 days. Apoptotic cells showed caspase-3/7 activity with green fluorescence. The data are expressed as the mean ± SD. * p < 0.01 vs. Cont si RNA; # p < 0.01. n = 5 for each group. (Con-Con si , 0.2 ± 0.3; Con-Ifit3 si , 0.4 ± 0.64; Dep-Cont si , 0.7 ± 1.0; Dep-Ifit3 si , 24.1 ± 6.2; GCDC-Cont si , 0.1 ± 0.2; Dep-Ifit3 si , 8.7 ± 4.4). Scales are 10 μm. ( G ) Cellular senescence was effectively decreased in BECs with the induction of cellular senescence (serum depletion and GCDC) with a treatment with Ifit3 si RNA or control si RNA for 4 days. Cellular senescence was assessed by senescence-associated β-galactosidase activity (SA-β-gal) after treatment with serum deprivation (Dep) or GCDC (500 nM) and Ifit3 si RNA or control si RNA for 4 days. Percentage of cells positive for SA-β-gal was significantly increased in cells treated with Dep (SA-β-gal labeling index, 40.6 ± 6.8) or GCDC (33.7 ± 5.8). Treatment with Ifit3 si RNA significantly decreased cellular senescence in each condition (Dep + Ifit3 si RNA, 29.2 ± 6.0; GCDC + Ifit3 si RNA (9.9 ± 3.1). Data was expressed as mean ± SD. * p < 0.01 compared to control + Cont si RNA. # p < 0.01, ** p < 0.05. The data are expressed as the n = 5 for each group. Scales are 10 μm.

Article Snippet: Validated si RNA for Ifit3 and negative control si RNA were purchased from Santa-Cruz biotech (Santa-Cruz, CA, USA) and QIAGEN, respectively.

Techniques: Knockdown, Serum Depletion, Expressing, Small Interfering RNA, Control, Western Blot, Activity Assay, BrdU Staining, WST Assay, Cell Cycle Assay, Fluorescence, Labeling