Journal: Nature Communications

Article Title: Primary cilia and SHH signaling impairments in human and mouse models of Parkinson’s disease

doi: 10.1038/s41467-022-32229-9

Figure Lengend Snippet: a Immunostainings exemplarily shown for O3H-R1-003. hiPSC pluripotency staining for markers OCT4, NANOG, SOX2. Scale bar=200 µm. hNPC staining for markers SOX1, SOX2, NESTIN, PAX6. Scale bar=100 µm. Neuron staining for markers TUBB3 and DAn marker TH. Scale bar=100 µm. Astrocyte staining for markers GFAP and SLC1A3. Scale bar=100 µm. b Summary of somatic CNVs identified in hNPC clones by chromosomal microarray analysis shown as total number of somatic CNVs detected per analyzed clone and average length of CNVs (in kb; green=copy number gain; orange=copy number loss) per analyzed clone. n = 5 Ctrl and 7 sPD patients. c Circos plot showing the genomic distribution of somatic CNVs in Ctrl (blue) and sPD (red) clones. d Quantification of RBFOX3 (synonym: NeuN) positive as well as TH / RBFOX3 double-positive cells in DAn populations. n = 5 Ctrl and 7 sPD clones, in triplicates. e Characterization of neurite morphologies of DAns. Boxplots show the average number of neurites emerging from TH positive cell bodies, their average number of branch points and their average length. n = 5 Ctrl and 7 sPD clones, in triplicates. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value. Each dot represents one patient. P -values were determined by two-sided t -test d (right), e ; two-sided Mann–Whitney-U test b, d (left). * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were diluted as follows: AC-TUB (T6793, Sigma-Aldrich; 1:1000), ARL13B (17711-1-AP, Proteintech; 1:500), GFAP (MAB360, Millipore; 1:250), GLI3 (AF3690, R&D; 1:100), NANOG (AF1997, R&D Systems; 1:200), NES (Ma1110, Thermo Fisher Scientific; 1:250), PAX6 (Ab78545, Abcam; 1:200), PITX3 (38-2850, Invitrogen; 1:300), POU5F1 (2840 S, Cell Signaling; 1:500), RBFOX3 (ab104224, Abcam; 1:800), SLC1A3 (NB100-1869, Novus Biologicals; 1:250), SMO (sc166685, Santa Cruz; 1:500), SOX1 (Ab87775, Abcam; 1:500), SOX2 (sc17320, Santa Cruz; 1:500), TH (P40101, PelFreez; 1:600), TUBB3 (T5076, Sigma-Aldrich; 1:1000).

Techniques: Staining, Marker, Clone Assay, Microarray, Full Display Name, MANN-WHITNEY

Journal: The Journal of Pathology

Article Title: Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells

doi: 10.1002/path.4439

Figure Lengend Snippet: Double minute chromosome (DM)-containing tumour cells often show constitutive activation of the ERK1/2–MAPK pathway. (A, F) The number of DMs/cell in various human malignant tumour cells (bars represent mean ± SD). (B–D, G) Phosphorylation status of ERK1/2, p38 and JNK1/2/3 in DM-containing tumour cells; phosphorylation of ERK1/2, p38 and JNK1/2/3 in MEKK3- or p38-over-expressing or TNF α -treated HEK293T cells (10 ng/ml for 30 min) are shown as positive controls. (E) Microarray analysis of UACC-1598DM compared to UACC-1598HSR cells

Article Snippet: Recombinant human TNF α was purchased from BioVision (Mountain View, CA, USA).

Techniques: Activation Assay, Expressing, Microarray

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Upregulation of CXCL1 in DCs isolated from mice and patients with CRC. Increased CXCL1, CXCL2 and CXCL3 ( A ) in SW620-conditioned TADCs, as determined by microarray. Elevated CXCL1 in SW620-conditioned TADCs at mRNA ( B ) and protein ( C ) levels. TADCs were generated by culturing CD14 + monocytes with RPMI, L-15 medium (50%), and SW620-CM (50%) and presenting in GM-CSF (10 ng/mL) and IL-4 (10 ng/mL) for five days. The expressions of mRNA and protein were assessed by microarray, qRT-PCR and Luminex assays. ( D ) The level of CXCL1 in the DCs isolated from patients with CRC. CD11c + cells were isolated from healthy donors and patients with CRC, and the levels of CXCL1 were measured by Luminex assay. ( E ) The levels of CXCL1 in DCs isolated from colon cancer-bearing mice. Mouse colon cancer CT26 cells were injected into mice via intraperitoneal injection. After 14 days, peritoneal lymph nodes were harvested. CD11c + DCs were isolated from the lymph nodes, and the culture medium collected after 24 h incubation. CXCL1 levels were determined by ELISA. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Isolation, Microarray, Generated, Quantitative RT-PCR, Luminex, Injection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 increased cancer stem cell (CSC) properties in SW620 colon cancer cells. Anchorage-independent growth ( A ) and tumor spheroid formation ( B ) of CXCL1-treated SW620 cells. The expression of CD44, CD326 and CD133 ( C ) and aldehyde dehydrogenase (ALDH) activity ( D ) in tumor spheroids. SW620 cells were cultured with CXCL1 protein in soft agar (0.4%) or ultra-low attachment wells for 40 and 10 days, respectively. The tumor spheroids were counted by microscope. ALDH activity and surface markers of tumor spheroids were determined by ALDEFLUOR assay and flow cytometry. ( E ) Level of CSC-related transcription factors in tumor spheroids. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Microscopy, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: CXCL1 enhanced cancer progression in SW620 cells. CXCL1 increased cell migration, as determined by wound healing ( A ) and transwell ( B ) analysis. The migration ability of SW620 cancer cells was assessed by wound healing assay and transwell assay. CXCL1 acted as a chemoattractant for cancer migration in the transwell system for 24 h. The effect of CXCL1 on the expression of MMP-7 ( C ), EMMPRIN ( D ) and epithelial-to-mesenchymal transition (EMT) ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the levels of various MMPs and EMT markers were determined by Luminex assay and Western blot. Results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Luminex, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Gene Profile of CXCL1-Treated SW620 Cells.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The correlation of CXCL1-regulated genes with CRC patient outcome. The correlation of overall survival ( A ), relapse-free ( B ) and metastasis-free rates ( C ) with CXCL-1 regulated genes. ( D ) The effect of CXCL1 in the expression of PTHLH and TCF4 at mRNA levels. CXCL1 increased the expression of PTHrP ( E ) in SW620 cells. SW620 cells were treated with CXCL1 for 24 h, and the expressions of PTHLH and TCF4 were determined by qRT-PCR and ELISA, respectively. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*).

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: The effect of CXCL1 on the expression of microRNAs (miRNAs) in colon cancer. ( A ) The regulatory effect of CXCL1 on the expressions of miRNAs in colon cancer. Prognostic significance of miR-105 and miR-597 in overall survival ( B ), relapse-free ( C ) and metastasis-free rates ( D ) in colon cancer patients. The effect of CXCL1 on the levels of miR-105 ( E ) and miR-597 ( F ) in colon cancer. SW620 cells were treated with CXCL1 for 24 h, and the expression of miR-105 and miR-597 determined by qRT-PCR. Each value is the mean ± SD of three determinations. * Significant difference between the two test groups ( p < 0.05) (*). Hsa, Homo sapiens; TCGA, The Cancer Genome Atlas; HR, hazard ratio; PVAL, p -value.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Tumor-Associated Dendritic Cells and Colon Cancer Cells Contributes to Tumor Progression via CXCL1

doi: 10.3390/ijms19082427

Figure Lengend Snippet: Proposed model of TADCs in colon cancer. Soluble factors in colon cancer cause abnormal phenotypes of dendritic cells, which in turn increase cancer stem cell properties, cell mobility, and EMT of colon cancer by producing the inflammatory chemokine CXCL1. Transcriptome analysis indicates that CXCL1 increases potential oncogene expression in colon cancer, including PTHLH , TYRP1 , FOXO1 , TCF4 , ZNF880 and the onco-miR miR-105. Our study indicates a new mechanism by which the colon cancer milieu exploits DC plasticity to support cancer progression.

Article Snippet: Recombinant human CXCL1 protein was obtained from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Immunity

Article Title: Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

doi: 10.1016/j.immuni.2019.08.017

Figure Lengend Snippet:

Article Snippet: Recombinant Human/Mouse/Rat BMP-2 Protein , RnD Systems , Cat#355-BM-010.

Techniques: Control, Recombinant, Blocking Assay, Irradiation, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Microarray, Software, Microscopy

Journal: Frontiers in Immunology

Article Title: RNA-Seq Analysis of IL-1B and IL-36 Responses in Epidermal Keratinocytes Identifies a Shared MyD88-Dependent Gene Signature

doi: 10.3389/fimmu.2018.00080

Figure Lengend Snippet: Type I and II interferon genes have time-dependent IL-1B and IL-36 responses. (A) Interferon gamma receptor 1 ( IFNGR1 ). (B) Interferon gamma receptor 2 ( IFNGR2 ). (C) Interferon alpha and beta receptor subunit 2 ( IFNAR2 ). In panels (A–C) , average FPKM (±1 SE) is shown for each group, and asterisks denote significant differences relative to the control (CTL) treatment at the corresponding time point (paired two-sample t -test; n = 2 or 3 per treatment). (D) Genes with interferon response factor 1 (IRF1) binding sites (5 kb upstream region). The middle 50% of FC estimates is shown for genes with 2+ IRF1 binding sites compared with genes with fewer IRF1 sites (magenta font, horizontal axis: P < 0.05, Wilcoxon rank sum test). The IRF1 position weight matrix is shown (right) along with the IRF1 tetrameric structure (bottom right; NCBI structure database). (E) IFN-induced gene signature scores. IFN-induced genes were identified from microarray studies of IFN-treated keratinocytes (left margin), and the average FC for these genes was calculated in IL-1B/IL-36 experiments (bottom margin). Left margin labels indicate the cytokine concentration (in ml), treatment duration, and GEO series accession number. All cytokine experiments were replicated with at least two samples per treatment. (F) Top 30 IFN-g-induced genes (identified from GSE36287). (G) Top 35 INFa-induced genes (identified from GSE36287). (H) Self-organizing maps (SOMs). The SOM layout was determined only from IFN-g-induced genes (i.e., 2,500 genes most strongly induced by IFN-g, GSE36287). Colors reflect average FC estimates for IFN-g-induced genes assigned to each SOM region (columns 1 and 2 on left). The final column (yellow–blue) displays the mean FC difference for each cytokine with respect to each SOM region (8 h mean FC–24 h mean FC; log 2 scale).

Article Snippet: MYD88-KO KCs including WT KCs were grown in 12-well plates, and cells were treated with recombinant IL-1 beta (10 μg/ml; R&D Systems # 201-LB-025), IL-36 gamma (10 μg/ml; R&D Systems # 6835-IL-010), IFN-gamma (50 μg/ml; R&D Systems # 285-IF-100), IL-17A (20 μg/ml; R&D Systems # 317-ILB-050), and/or TNF-alpha (10 μg/ml; R&D Systems # 210-TA-005) for 8 or 24 h. RNAs were isolated from cell cultures using Qiagen RNeasy plus kit (Cat # 74136).

Techniques: Control, Binding Assay, Microarray, Concentration Assay

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Antibody panels used for immunohistochemistry on HD tissue microarrays.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemistry, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Immunohistochemical profiling of HTT inclusion body ubiquitination and associated triage protein binding in the HD human middle temporal gyrus. Multiplexed immunohistochemical approaches were used to identify HTT inclusion bodies, ubiquitin species, and triage proteins in neurologically normal and HD human middle temporal gyrus tissue microarray cores. Example images from HD case, HC150, are shown. HTT inclusion body antibodies, EM48 ( A ), EPR ( B ), and MW1 ( C ), were used for labelling together with antibodies for pan-ubiquitin ( D ), K48- and K63-linked polyubiquitination ( E and F ), p62 ( G ), and ubiquilin 2 ( H ), with a Hoechst nuclear counterstain ( I ); scale bars = 20 μm.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemical staining, Ubiquitin Proteomics, Protein Binding, Microarray

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: HTT inclusion bodies are not frequently ubiquitinated, but when ubiquitinated, are predominantly ubiquitinated by K63-linked ubiquitin. Immunohistochemical labelling revealed that EM48, EPR, and/or MW1 HTT inclusion bodies were ubiquitinated by K48- and/or K63-linked ubiquitin ( A ); a representative image of K48- and K63-ubiquitinated HTT inclusion bodies from HD case, HC145, is shown; scale bars = 10 μm. The ubiquitination status of each HTT inclusion body was determined by labelling for pan-, K48-, and K63-linked ubiquitin, where positive labelling was identified if the maximum intensity was above manually determined thresholds. The percentage of EM48 + versus EM48- ( B ), EPR + versus EPR- ( C ), and MW1 + versus MW1- ( D ) HTT inclusion bodies that were ubiquitinated (either pan, K48-, and/or K63-linked) were compared using a Wilcoxon matched-pairs signed rank test. The percentage of ubiquitinated HTT inclusion bodies was determined for each EM48, EPR, and MW1 +/- phenotype per HD case ( E ), and compared between phenotypes using a mixed-effects analysis, with Geisser-Greenhouse correction and Tukey’s multiple comparisons test. The percentage of ubiquitinated HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin was compared using a Wilcoxon matched-pairs signed rank test ( F ). The percentage of ubiquitinated EM48 + versus EM48- ( G ), EPR + versus EPR- ( H ), and MW1 + versus MW1- ( I ) HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin were compared using an ordinary two-way ANOVA with Tukey’s multiple comparisons test. The percentage of EM48, EPR, and MW1 +/- immunophenotypes HTT inclusion bodies identified as being ubiquitinated by K48- or K63-linked chains were compared using an ordinary two-way ANOVA with Sidak’s multiple comparisons test ( J ). Data are presented as truncated violin plots ( n = 20). Statistical significance of differences shown for B-D and F-J: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Statistical significance for E is shown in Supplementary Table 3.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Summary of HTT inclusion body characteristics. Heatmap organised by HTT inclusion body phenotype, with each column representing a single case and each bar coloured according to that case’s value for the characteristic outlined by the row title ( A ). Schematic illustrating the general characteristics of each HTT inclusion body phenotype: (1) EPR + MW1 + inclusion bodies are more frequently located in the nucleus compared to other phenotypes, (2) HTT inclusion bodies that label for more than one epitope-specific antibody are more frequently ubiquitinated, and that ubiquitination occurs more frequently by K63- compared to K48-linked ubiquitin chains, (3) Ubiquitinated HTT inclusion bodies are more frequently tagged by ubiquilin 2 than p62 ( B ). Schematic summarising our hypothesis of HTT inclusion body immunophenotype, ubiquitination, and triage protein tagging with increasing HD severity ( C ); created in BioRender.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics