Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: Analysis of OXTR expression in various tumor tissues in TCGA. (a) Transcriptional levels of OXTR in different tumor tissues and adjacent normal tissues. (b) The relationships between OXTR expression levels in various tumors and OS were analyzed by KM curve. COAD: low OXTR group: n = 68, high OXTR group: n = 68; LUAD: low OXTR group: n = 112, high OXTR group: n = 119; STAD: low OXTR group: n = 94, high OXTR group: n = 95. ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: esophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; HPV: human papillomavirus; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LAML: acute myeloid leukemia; LGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; MESO: mesothelioma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumors; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; UCS: uterine carcinosarcoma; UVM: uveal melanoma; LumA: luminal A; LumB: luminal B; Her2: human epidermal growth factor receptor-2. **: p < 0.01; ***: p < 0.001.

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques: Expressing

Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: OXTR mRNA level is high in COAD patients from six datasets. (a–f) GraphPad Prism 8 was used to analyze the expression of OXTR in COAD samples from TCGA (a), GSE9348 (b), GSE32323 (c), GSE38026 (d), GSE44076 (e), and GSE115313 (f) databases. ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques: Expressing

Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: Association between OXTR expression and the clinical parameters in patients with COAD in TCGA

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques: Expressing

Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: ROC curve analysis of OXTR expression related to the indicated clinical parameters in COAD patients. (a) The ROC curve analysis of COAD tissue and unpaired normal tissue. (b) The ROC curve analysis of COAD tissues and paired normal tissues. (c–f) ROC curve analysis of TNM stage (c), T stage (d), OS (e), and living status (f). AUC: area under the curve; OS: overall survival.

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques: Expressing

Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: COAD patients with high levels of OXTR are more likely to show short OS time. (a) The correlation analysis between OXTR mRNA level and OS in COAD patients was performed by the KM curve. (b–k) KM curve analysis of male (b), female (c), age > 60 years (d), age ≤ 60 years (e), stages T1 + T2 (f), stages T3 + T4 (g), no metastasis (h), N0 stage (i), stages I + II (j), and stages III + IV (k) patients with COAD.

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques:

Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: KEGG enrichment analysis of the correlation of OXTR mRNA level with colorectal cancer and four main signaling pathways in colorectal cancer. (a) Gene set enrichment analysis of the correlation between OXTR expression level and the expression of genes related to four signaling pathways in colorectal cancer. (b) Correlation analysis between the OXTR expression level and mRNA levels of genes related to four signaling pathways in COAD. KEGG: kyoto encyclopedia of genes and genomes.

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques: Protein-Protein interactions, Expressing

Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: Correlations between OXTR expression and immune infiltration in COAD. (a) Rod diagram of the relationship between OXTR levels and infiltration levels of various immune cells. (b) Scatter plot of the association between OXTR levels and infiltration levels of type 2T helper cell, central memory CD8 T cell, CD57 bright natural killer cell, activated CD8 T cell, activated B cell, and Type 1T helper cell.

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques: Expressing

Journal: Open Medicine

Article Title: Prognostic role of oxytocin receptor in colon adenocarcinoma

doi: 10.1515/med-2021-0387

Figure Lengend Snippet: Silencing OXTR inhibits cell proliferation, migration, and invasion. (a and b) OXTR mRNA (a) and protein (b) levels in human normal colonic epithelial cell line (NCM-460) and COAD cell lines (HCT-8, SW480, SW620, and RKO). (c and d) After HCT-8 and SW480 cells were transfected with siOXTR, qPCR (c) and western blotting (d) assays were used to analyze the levels of OXTR mRNA and protein in the cells, respectively. (e‒h) After silencing OXTR, MTT assay, PI staining, and transwell assay were used to detect cell proliferation (e), cell cycle (f), migration (g), and invasion (h). siOXTR: small interfering RNA of OXTR; siNC: the negative control of siOXTR; PI: propidium iodide. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The human COAD cell lines HCT-8 (catalog number: IM-H099) and RKO (catalog number: IM-H409) were cultured in RPMI-1640 Medium (ATCC, Catalog No. 30-2001) with 10% horse serum and Eagle’s Minimum Essential Medium with 10% fetal bovine serum (FBS), respectively, and SW620 (catalog number: IM-H112) and SW480 (catalog number: IM-H111) were cultured in Leibovitz’s L-15 Medium (ATCC, Catalog No. 30-2008) with 10% FBS.

Techniques: Migration, Transfection, Western Blot, MTT Assay, Staining, Transwell Assay, Small Interfering RNA, Negative Control

Journal: BMC Microbiology

Article Title: Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni

doi: 10.1186/1471-2180-9-220

Figure Lengend Snippet: Analyses of biological activities of CDT . (A) Cytolethal distending effect by OMVs on HCT8 cells. HCT8 cells without treatment (a, b), HCT8 cells treated with 50 μl of OMVs (total protein concentration was 60 μg/ml) from wild type C. jejuni strain 81-176 (c, d), or from the cdtA ::km mutant (e, f). After 72 hours of treatment the actin filaments and nuclei were stained with phalloidin and DAPI, respectively, as described in materials and methods. Upper panels (a, c, e) show merged images from staining with both dyes and lower panels (b, d, f) show images from DAPI staining only. Bars represent 40 μm. (B) Effect of thymidine uptake on HCT8 cells after treatment with OMVs from wild type C. jejuni strain 81-176 and the cdt::km mutant strain DS104 for 48 h. Cells were grown in 96-well plates and 10 μl of OMVs were added to the wells. The results are from triplicate wells and two independent experiments. Data are expressed as mean percentage (± SE).

Article Snippet: The human ileocecum carcinoma cell line HCT8 (ATCC number CCL-224) was kindly provided by the Institute for Molecular Infection Biology, University of Würzburg.

Techniques: Protein Concentration, Mutagenesis, Staining

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: IL-1β–induced INAVA puncta are biomolecular condensates. (A) Time course of condensate formation in HCT8-INAVA-GFP cells treated with IL-1β. Scale bar = 5 μm. (B) Quantification of puncta per 1,000 cells, mean ± SD, n = 2. Images acquired at four positions/well. (C) As in B but analyzed by puncta area. (D) HCT8 cells expressing the long or short isoform of INAVA treated with IL-1β for 90 min. Scale bar = 10 μm. (E) IL-1β–induced puncta fusion over time in HCT8 cells. Scale bar = 2 μm. (F) FRAP of IL-1β–induced puncta at 30 min. Scale bar = 2 μm. (G) FRAP of “young” (30 min) and “old” (90 min) puncta, mean ± SEM, n = 10. (H) Velocity tracking of puncta, mean ± SEM, n = 36. ****, P < 0.0001.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: Description and validation of the screen for IL-1β–induced INAVA condensates. (A) Predictor of Natural Disordered Regions (PONDR) score displaying intrinsic disordered and ordered regions of the long and short isoforms of INAVA. (B) INAVA does not colocalize with lysosomes or endosomes. IL-1β–induced HCT8-INAVA-GFP cells were treated with transferrin-Alexa Fluor 647 (10 μg/ml) for 30 min as a marker for endosomes. Cells were washed, fixed with 4% PFA, and stained with α-LAMP1 as a marker for lysosomes. Images were taken using confocal microscopy. Scale bar = 10 μm. (C) Table of small compound libraries screened in our study. (D) Representative image of the automated analysis used for INAVA-GFP puncta quantification. Hoechst (nuclei). Scale bar = 10 μm. (E) Automated Z-factor for IL-1β– and H 2 O 2 -induced INAVA-GFP puncta screens. EMD, EMD Biosciences; FDA, US Food and Drug Administration; GPCR, G-Protein Coupled Receptor; L, Longwood; LINCS, Library of Integrated Network-Based Cellular Signatures; NIH, National Institutes of Health.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Marker, Staining, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: A small-molecule approach to delineate the biology of INAVA condensates. (A) Schematic (left panel) and results (middle panel) of screen for inducers of INAVA-GFP puncta. Ranked Z-scores are shown on the right panel; several highly ranked HSP90 inhibitors (red) or ROS inducers (blue) are indicated. Frequencies of distribution of negative controls are displayed to the right of graph. (B) Schematic (left panel) and results (middle panel) of screen for compounds that block IL-1β–induced puncta. Ranked Z-scores are shown on the right panel; a high proportion of top hits were MAPK p38α (red) and mTOR (blue) inhibitors. Frequencies of distribution of negative controls are displayed to the right of graph.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Blocking Assay

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: ROS and HSP90 and proteasome inhibitors induce INAVA puncta. (A and B) Dose response for HSP90 inhibitors (A) ganetespib and (B) 17AAG, mean ± SEM, n = 3. (C) Time course of puncta formation by IL-1β (10 ng/ml), ganetespib (1 μM) and 17AAG (10 μM), mean ± SD, n = 2. (D) Time course of puncta formation and resolution after treatment with agonists noted above, n = 2. (E) Time course images from D. Scale bar = 10 μm. (F and G) Confocal images of HCT8-INAVA-GFP cells stained for (F) vimentin or (G) G3BP1 following treatment with IL-1β or H 2 O 2 for 90 min. Hoechst (nuclei). Line scans (arrows) display relative fluorescence intensity of INAVA and (F) vimentin or (G) G3BP1. Scale bar = 20 μm.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: Characterization of INAVA puncta inducers. (A and B) Dose–response curves of (A) HSP90 inhibitors or (B) proteasome inhibitors. Images were taken by high-content imaging at 20× magnification with four positions/well, mean ± SEM, n = 3. (C) Representative images from A and B. Scale bar = 10 μm. (D) Representative images from ROS inducers identified in the INAVA puncta inducer screen. Scale bar = 10 μm. (E) HCT8 cells stably expressing only GFP were treated with corresponding compounds for 90 min, fixed, and imaged. Scale bar = 40 μm. (F) HCT8-INAVA-GFP cells treated with IL-1β (10 ng/ml), H 2 O 2 (1 mM), or ganetespib (1 μM) for the early and late time points indicated. Cells were fixed and stained with vimentin. Scale bar = 20 μM. (G) INAVA protein–protein interactors annotated in BioGRID ( https://thebiogrid.org ). Dashed circles highlight interactors, including cytohesin family members (blue) and multisubunit ubiquitin E3 complexes (green). The multisubunit dynactin complex (red) involved in the aggresome pathway was also identified. (H and I) HCT8-INAVA-GFP cells were treated with (H) IL-1β (10 ng/ml), ganetespib (1 μM), MG132 (10 μM), or (I) H 2 O 2 (1 mM) for 90 min. Cells were fixed and stained with AMYLO-GLO. Scale bar = 20 μM. (J) HCT8-INAVA-GFP cells were treated with IL-1β (10 ng/ml) or H 2 O 2 (1 mM) for 90 min and stained with EDC4 (left panel). Fluorescent line scans (arrow) of INAVA (green) and EDC4 (red) are displayed (right panel). Scale bar = 10 μm. (K) HCT8-INAVA-GFP cells were treated with IL-1β (10 ng/ml), ganetespib (1 μM), or H 2 O 2 (1 mM) for 90 min and stained with HDAC6. Scale bar = 20 μm. (L) Pulse-chase experiments in HCT8 INAVA-GFP cells treated with IL-1β (10 ng/ml), H 2 O 2 (1 mM), or ganetespib (1 μM) for 30 min. Following treatment, cells were washed with complete media, chased, and analyzed at the time points indicated, mean ± SEM, n = 2. (M) HCT8-INAVA-GFP cells were treated with condensate inducers as above, including MG132 (10 μM) at the indicated time points. Cells were harvested and analyzed for Western blotting. Anti-HA measures HA tag present in INAVA-GFP, and actin was used as a loading control. (N) Same as D but treated with ganetespib (1 μM and 300 nM). Samples were collected at 1.5 h and 20 h for Western blot analysis. The unit of measure for the Western blots is kilodaltons.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Imaging, Stable Transfection, Expressing, Staining, Pulse Chase, Western Blot

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: INAVA condensates as a mediator of cellular proteostasis. (A) HCT8-INAVA-GFP cells were treated with IL-1β (10 ng/ml) for 1 h and ganetespib (1 μM) and 17AAG (10 μM) for 1.5 h. Cells were fixed and stained with conjugated ubiquitin antibody FK2. Scale bar = 10 μm. (B) HCT8-INAVA-GFP cells expressing doxycycline-inducible myc-βTrCP2 were treated with doxycycline (1 μg/ml) overnight followed by the condensate inducers as in A or H 2 O 2 (1 mM) for 90 min. Cells were fixed and stained with α-myc for βTrCP2. Scale bar = 10 μm. (C) HEK293T cells were transfected with HA-ubiquitin with stably expressed empty vector (EV), GFP-INAVA long or short isoform, and a CUPID domain mutant (C141A). Cells were treated with or without H 2 O 2 (1 mM) for 90 min. Whole-cell lysates were collected and analyzed by immunoblot for HA (ubiquitin), GFP (INAVA), and actin. The unit of measure for the Western blots is kilodaltons.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Staining, Expressing, Transfection, Stable Transfection, Plasmid Preparation, Mutagenesis, Western Blot

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: H 2 O 2 -induced INAVA condensates colocalize with conjugated ubiquitin: control experiments for βTrCP2 colocalization. (A) HCT8-INAVA-GFP cells were treated with H 2 O 2 (1 mM) for 90 min and stained with conjugated ubiquitin antibody FK2. Scale bar = 10 μm. (B) Control images for of cells expressing only INAVA-GFP. HCT8-INAVA-GFP cells expressing doxycycline (Dox)-inducible myc-βTrCP2 without Dox treatment. Cells treated with condensate inducers as in . Cells were fixed and stained with phalloidin-TRITC (F-actin) and α-myc. (C) HCT8 cells stably expressing Dox-inducible myc-βTrCP2. Cells were treated overnight with Dox (1 μg/ml), fixed, and stained with phalloidin-TRITC and α-myc. Scale bar = 10 μm.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Staining, Expressing, Stable Transfection

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: Validated p38α and mTOR inhibitors discovered in the screen, characterization of H 2 O 2 -induced INAVA puncta as biomolecular condensates, and schematic of the screen for puncta inhibitors. (A–C) Dose–response curves of all (A) p38α inhibitors, (B) mTOR inhibitors, and (C) translation inhibitors validated from the IL-1β–induced INAVA puncta inhibition screen. Images were taken by high-content imaging at 20× magnification at four positions/well, mean ± SEM, n = 3. (D) Representative images from dose–response experiments (p38α inhibitors [c–m]: p38α inhibitor VIII, PD169316, RWJ-67657, LY2228820, VX702, SB202190, SB239063, PH-797804, GW856553X, SB242235, SB220025; mTOR inhibitors [n–q]: GDC-0980, BEZ235, KU0063794, PI-103). Hoechst (nuclei). Scale bar = 10 μm. (E) HCT8-INAVA-GFP cells were pretreated with INK128 (10 μM) or SB203580 (10 μM) for 30 min followed by treatment with IL-1β for 90 min. Cells were harvested and analyzed by Western blotting. Anti-HA detects INAVA-GFP, and actin was used as loading control. (F) HCT8-INAVA-GFP cells were treated with 3% and 5% 1,6-hexanediol (HD) for 5 and 15 min and were analyzed for INAVA-GFP puncta, mean ± SD. (G) Same as F but the time point at 30 min with 3% 1,6-HD, n = 3. (H and I) NF-κΒ luciferase reporter assay with (H) p38α inhibitor SB203580 or (I) mTOR inhibitor INK128. HEK293T cells transfected with empty vector (EV) or INAVA, NF-κΒ firefly luciferase and SV40- R. reniformis were pretreated with SB203580 or INK128 for 1 h and then treated with IL-1β (10 ng/ml) for 4 h and assayed for luminescence. Each point represents average luminescence for all replicates within each experiment, N = 49 per condition, four independent experiments performed, mean ± SD. (J) HCT8-INAVA-GFP cells expressing doxycycline-inducible myc-TRAF6. Cells were treated overnight with doxycycline (1 μg/ml) and then treated with IL-1β (10 ng/ml) for 60 min. Cells were fixed, stained with α-myc to detect myc-TRAF6 and processed by spinning disk confocal imaging. Scale bar = 10 μm. (K) FRAP of H 2 O 2 -induced INAVA-GFP “young” (45 min; n = 6) and “old” (120 min; n = 5) puncta in HCT8-INAVA-GFP cells. Bleaching was performed at the indicated time point (arrow), and recovery was allowed to occur at 37°C in 5% CO 2 , mean ± SEM. (L) Velocity tracking of H 2 O 2 -induced INAVA-GFP puncta formed at different time points, mean ± SEM, n = 36. (M) Schematic of screen to identify inhibitors of H 2 O 2 -induced INAVA-GFP puncta (left panel) in HCT8 cells. Shown are ranked Z-scores of hits (right panel) and relative ranking of select compounds of interest: inhibitors of protein translation (green), MAPK p38α (red), or mTOR (blue). Frequencies of distribution of negative controls are displayed to the right of the graph. (N) HCT8-INAVA-GFP cells were pretreated with cycloheximide (CHX; 10 μM) or MELK inhibitor OTSSP167 (10 μM) for 30 min followed by IL-1β (10 ng/ml) for 1 h. Cells were harvested and analyzed for Western blotting. Anti-HA measures HA tag present in INAVA-GFP, and actin was used as a loading control. ****, P < 0.0001. The unit of measure for the Western blots is kilodaltons.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Inhibition, Imaging, Western Blot, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: Inhibitors of the MAPK p38α and mTOR pathway induce the resolution of INAVA condensates. (A and B) In HCT8-INAVA-GFP cells, IL-1β– and H 2 O 2 -induced INAVA-GFP condensates are blocked by mTOR inhibitors (A) and p38α inhibitors (B). Graphs display results from high-content imaging, mean ± SD, n = 2. (C and D) Dose–response curves for (C) mTOR inhibitor INK128 or (D) p38α inhibitor SB203580, mean ± SEM, n = 3. (E) High-content images from dose–response studies. HCT8-INAVA-GFP cells were pretreated with INK128 (43 μM) or SB203580 (14 μM) for 30 min followed by IL-1β (10 ng/ml) for 90 min. Scale bar = 5 μm. (F) Cells were treated with IL-1β (10 ng/ml) for 50 min followed by INK128 (10 μM) or SB203580 (10 μM) then fixed and stained with Hoechst (nuclei) at indicated time points, n = 2 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Imaging, Staining

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: Blockade of INAVA and FUS, FMR1, and FXR1 condensates. (A) HeLa cells stably expressing FUS-GFP were pretreated with OTSSP167 (5 μM), INK128 (10 μM), or SB203580 (10 μM) for 30 min followed by sodium arsenate (1 mM) for 90 min. Images were acquired at six positions/well at 20× magnification, mean ± SD, n = 6. Reproduced in three independent studies. (B and C) As in A but with WT HeLa cells treated with MG132 (10 μM) for 5 h. Cells were fixed and stained with (B) α-FMR1 or (C) α-FXR1. (D) FUS-GFP images from A. (E) Images of FMR1 and FXR1 from B and C. Scale bars = 10 μm. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Stable Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: INK128 and OTSSP167 block FUS-GFP puncta; membrane and cytosolic localizations of INAVA are competing reactions. (A) HeLa FUS-GFP cells were treated with sodium arsenate (1 mM) for 90 min to allow FUS-GFP puncta formation. Cells were then treated with INK128 (10 μM) or OTSSP167 (10 μM). Cells were fixed at indicated time points and analyzed, mean ± SEM, n = 5. (B) INAVA-GFP localize to the membrane in Caco2BBe cells stably expressing INAVA-GFP (Caco2BBe-INAVA-GFP). Cells treated with IL-1β (10 ng/ml) for 1 h form INAVA-GFP puncta. Phalloidin-TRITC (actin). (C) Caco2BBe-INAVA-GFP cells were treated with ganetespib (1 μM), 17AAG (10 μM), or H 2 O 2 (1 mM) for 90 min and MG132 (10 μM) for 2 h. Fixed cells were stained with DRAQ5 (nuclei). Scale bar = 10 μm. (D) HCT8-INAVA-GFP cells were pretreated with Y27632, Y3998, and blebbistatin (33 μM for each compound) for 180 min. Cells were then treated with IL-1β (10 ng/ml) for 90 min, fixed, and imaged. Hoechst (nuclei). Scale bar = 20 μm.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Blocking Assay, Stable Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Small-molecule modulators of INAVA cytosolic condensate and cell–cell junction assemblies

doi: 10.1083/jcb.202007177

Figure Lengend Snippet: ROCK inhibitors rescue epithelial morphology in HCT8 cells. (A) Confocal images of HCT8-INAVA-GFP cells treated with ROCK inhibitors Y27632 (10 μM), Y39983 (10 μM), or myosin II inhibitor blebbistatin (50 μM) for 1 h. Scale bar = 10 μm. (B) Quantification of INAVA-GFP localized to lateral membranes, n = 120–150 cells/condition. (C) Confocal images of HCT8 WT and HCT8-INAVA-GFP cells treated as in A and stained with tight junction marker ZO-1. Scale bar = 10 μm. (D) Brightfield images of HCT8 WT and HCT8-INAVA-GFP cysts grown in Matrigel ± Y27632 (10 μM). Overview scale bar = 500 μm. Enlarged image scale bar = 100 μm. (E) Relative distribution of HCT8 cyst morphology from D, n = 80–98 cysts/condition. (F) Dose response of ROCK inhibitor Y39983, Y27632, or blebbistatin (left to right, 33 μM, 6.7 μM, 1.3 μM, 0.3 μM) in blocking IL-1β–induced condensates, mean ± SEM, n = 2. (G) HCT8-INAVA-GFP cells with doxycycline-inducible GEF mutant myc-ARNO-(E156K). Cells were treated overnight with doxycycline (1 μg/ml), fixed, stained, and imaged at four positions/well at 40× magnification, n = 2. Scale bar = 10 μm. (H) Confocal images (left) and quantification (right) of puncta inhibition as in G but treated with IL-1β (10 ng/ml) for 60 min. Scale bar = 20 μm, mean ± SEM, n = 2. *, P < 0.05; **, P < 0.01.

Article Snippet: HCT8 cells overexpressing INAVA-GFP (HCT8-INAVA-GFP) were grown in media (DMEM, 10% FBS, 5% penicillin/streptomycin) and plated at 6,250 cells/well on 384-well plates (781090; Greiner Bio-One) using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific).

Techniques: Staining, Marker, Blocking Assay, Mutagenesis, Inhibition