EMBO/SourceData huggingface dataset:
Figure 5Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).
Figure 6Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.
Figure 1A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.B, ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n=3; for NPC, n=7.C, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in training cohort.E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ). F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).
Figure 2A-C, Kaplan-Meier curves showing the overall survival (OS) curves of NPC patients with low or high INSL5 concentration (A), low (≤4000 copy/ml) or high (>4000 copy/ml) EBV DNA copy number (B), and the combination of INSL5 concentration with EBV DNA copy number (C).D-F, Kaplan-Meier curves showing the disease-free survival (DFS) curves of NPC patients with low or high INSL5 concentration (D), low or high EBV DNA copy number (E), and the combination of INSL5 concentration with EBV DNA copy number (F).
Figure 3A. Exogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2 and HK1 NPC cell lines. Scale bars represent 20μm.BMTT assay of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1.colony formation (C and D) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments.Brdu incorporation (E and F) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (E) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments. Scale bars represent 20μm in (E)migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (G) for migration assays migrated cells per field of view were plotted in H, respectively. The results are from three different experiments. Scale bars represent 100μm in (G)I, J, Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor size (I) n=11 mice per group.Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor weight of two groups. n=11 mice per group.
Figure 4A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively.C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis.D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines.E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity.H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells.basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells.HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells.L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.
Figure 5B, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min.C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μmD, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions.E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies.G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector.H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.
Figure 6A, Brdu assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 20μm.B, similar to (A), migration and invasion assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 100μm.C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.Tumor size (G) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.tumor weight (H) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.
Figure 1A Heatmap showing the unsupervised consensus clustering of TCGA melanomas (n=445: 356 metastases and 89 primary melanomas). The identified clusters are indicated below the heatmap: cluster 1 (n=55, red), cluster 2 (n=304, blue) and cluster 3 (n=86, green). Type of melanoma samples (primary or metastatic) and of metastatic site (subcutaneous, lymph node or distant) within each cluster are indicated below and above the heatmap, respectively. B Heatmap showing the unsupervised consensus clustering of TCGA primary melanomas (n=89) using the 1,000 most variable expressed genes in the same dataset. The identified signature clearly divides the TCGA dataset in two groups: Primary-A (n=34, red) and Primary-B (n=55, pink), as indicated. C Boxplots showing the distributions of the normalized rank-sums of the up-regulated (left panel) and down-regulated (right panel) genes in the identified signature. Down-regulated genes in Primary-B melanomas better discriminate the two groups of primary melanoma samples (p=5.9e-15, Wilcoxon rank-sum test). Median, minimum, maximum, 25th, 75th whiskers of ranksum distribution are shown.
Figure 2A Box and whiskers (from 5th to 95th percentile) plots depicting TINCR RNA levels in the TCGA SKCM RNA-seq dataset of melanoma samples. TINCR expression is higher in Primary A melanomas, and its reduction in Primary-B melanomas and all groups of metastatic samples is statistically significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.B Scatter-plot showing correlation between the expression of TINCR RNA and normalized rank-sums s of the down-regulated (upper panel) and up-regulated (lower panel) genes in the primary melanoma signature. TINCR expression strongly correlates (Spearman's correlation coefficient of 0.79, Spearman rank p-value: **p<0.01) with the expression of the down-regulated genes.C Dot plot showing qRT-PCR analysis of TINCR expression in primary (8) and metastatic (8) melanoma samples from surgical specimens or patient-derived xenografts (4 primary and 8 metastatic PDXs). TINCR RNA levels relative to the L32P housekeeping gene are shown. Paired primary and metastasis PDXs are marked with an asterisk. The difference between primary and metastatic samples is significant (Mann-Whitney test, **p< 0.01). Median, minimum, maximum, 25th, 75th whiskers of expression values are shown.
Figure 3A qRT-PCR quantification of TINCR RNA expression in MMC70 and WM902B cell lines, independently infected with two shRNA targeting TINCR expression (shTINCR-1 and -2). Cells transduced with shScamble (shSCR) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) measured in three independent biological experiments are shown. In both cell lines TINCR knockdown efficiency is more than 70% with the two shRNAs. Statistical analyses were performed using Student t -test (**: p<0.01).B In vitro migration assay of MMC70 and WM902B cells transduced with shTINCR-1, shTINCR-2 or shSCR. Relative migration (mean±sd) from three independent experiments is indicated. Statistical analyses were performed using Student t -test (***: p<0.001).C In vitro cell proliferation of shSCR or shTINCR infected MMC70 and WM902B cells assayed 72h after infection at the indicated time points using CellTiter-Glo luminescent kit (Promega). Cell growth from three biological replicates is indicated as proliferation rate relative to control cells (mean±sd). The difference between control and TINCR-silenced samples at all time points is not significant (Student t-test).D Upper panel: in vivo growth of tumors derived from intradermal injection of shTINCR-2 or shSCR WM902B cells in NSG mice. 600,000 WM902B cells per mouse were inoculated. Tumor volumes (mean±sd) were measured 8 weeks after transplantation. Results from two independent experiments (four animals per group) are shown. Statistical analysis between control and TINCR-silenced groups was performed using the Student t -test (**=p<0.01). Lower panel: qRT-PCR analysis of TINCR RNA expression in tumors derived from in vivo transplant of shSCR and shTINCR WM902B cells (8 weeks after injection). TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from four biological replicates (animals) from both experiments are shown.E Drug-response curves showing increased resistance of shTINCR-WM902B cells after treatment with BRAF (Vemurafenib; from 10 to 10,000 nM) and MEK (Trametinib; from 4 to 2,000 nM) inhibitors. The shSCR- (control) and shTINCR-WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter-Glo luminescent kit (Promega). Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).G Western blot analysis of a panel of proteins that are specifically associated to the proliferative or invasive phenotype. Total protein lysates were extracted from shSCR- and shTINCR-WM902B cells (lane 1 and 2) or shSCR- and shTINCR-MMC70 cells (lane 3 and 4) 10 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control.
Figure 4A qRT-PCR analysis of TINCR expression (left) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full-length TINCR cDNA. PDX cells infected with empty vector (Empty) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from three biological replicates are shown. Statistical analyses were performed using Student t -test (***: p<0.001).B In vitro relative migration (mean±sd) from three independent experiments (biological replicates) is shown in MM13 and MM2 TINCR overexpressing (TINCR) compared to control (Empty) cells. Statistical analyses were performed using Student t -test (***: p<0.001).C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points (1-5 days) using CellTiter-Glo viability assay. Relative proliferation rate (mean±sd) of three independent experiments are shown.D Drug-response curves showing sensitization to MEK inhibitor Trametinib of MM13 (NRAS-mutant PDX) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells (BRAF-mutant PDX). The Empty (control) and TINCR expressing MM13 cells were treated with increasing concentrations of drug (ranging from 5 to 5000 nM) and cell viability was assayed after 72 hours by CellTiter-Glo viability assay. Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2, EGFR, AXL, PDGFRβ, SOX10, and MITF in TINCR-overexpressing MM13 and MM2 cells. Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control. This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression (8-10 folds).F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice. Four animals (200,000 cells/mouse) per group were used in each experiment. Tumor growth (mean±sd) was monitored weekly and tumor volume (mean±sd) measured at 4 weeks for primary tumors (originated in the site of injection, left panel) and at 8 weeks for spontaneous metastases which colonized lymph nodes (right panel). The results of two independent experiments are shown. Statistical analyses of control and TINCR-overexpressing groups from both experiments were performed using Student t -test (**: p<0.01).G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown. Hematoxylin-eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells. Metastasis formation was evaluated 8 weeks after transplantation into NSG mice. Pictures of representative lungs and livers infiltrated with metastases are shown in the inset. Scale bars: 200μm.
Figure 5A Western blot analysis of integrated stress response activation in WM902B and MMC70 cells. Cells were harvested 96 h after shRNA infection and western blot performed using the indicated antibodies.B Representative comparison of translational efficiency (TE) in control (shSCR) and shTINCR WM902B cells 72h post infection. The genes with statistically significant TE up- and downregulation (FDR< 0.1) are highlighted in red and blue respectively. ATF4 is circled among the upregulated genes.C Heatmap representing top translationally upregulated and down regulated genes in TINCR WM902B cells. The plotted values are the log2 fold TE change values in each biological replicate (rep1 and rep2) relative to control (shSCR).D Bar-graph representing ribosome protected fragments (RPF) normalized coverage (mean±sd) mapping to ATF4 CDS in control (shSCR) and TINCR (shTINCR) depleted WM902B cells from two biological replicates is shown.E UCSC genome browser tracks of ribosome footprint data illustrating RPF density distribution across ATF4 mRNA in shSCR and shTINCR WM902B cells.
Figure 6B Q-PCR validation of TINCR and ATF4 RNA recovery in TINCR antisense pulldown. Enrichment relative to input (mean±sd) from three independent pulldown experiments is shown. Statistical analyses were performed using Student t -test (**: p<0.01, ***: p<0.001).C UCSC genome browser tracks of TINCR-RIA-seq sequencing reads illustrating coverage and enrichment across ATF4 transcript in WM902B and MMC70 cells.
Figure 2(B) Cell viability of isolated cell clones and WT AN3-12 cells following 72h remdesivir treatment with the indicated doses. Mean ±SEM of 2-4 biological replicates is displayed.
Figure 3 (A) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with Human recombinant soluble ACE 2 (hrsACE2) (200µg/ml) and remdesivir (Remd. 4µM). Viral RNA level was determined at 15 hours after virus inoculation.  (B) Treatment of SARS-CoV-2 (106 PFU) infected human kidney organoids with hrsACE2 (200µg/ml) and/or remdesivir (Remd.4µM)). Viral RNA was determined by qRT-PCR 72 hours after the inoculation of 106 PFU of SARS-CoV-2.  (C) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM)).  (D) Treatment of SARS-CoV-2 (106 PFU) infected kidney organoids with hrsACE2 (10µg/ml) and remdesivir (4µM).  (E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi).
Figure 1(A , B) A brightfield image showing the zebrafish thyroid gland along with surrounding tissue. The thyroid follicles reside in the soft tissue surrounding the ventral aorta, which extends from the outflow tract of the heart into the gills towards the basibranchial cartilage in the lower jaw. The thyroid follicular cells, or thyrocytes, are labeled in green in the Tg(tg:nls-mVenus-NTR) transgenic line (B'). Scale bars: 500 µm(C) Maximum intensity projection of 3D confocal stack obtained from the dissected thyroid gland of Tg(tg:nls-EGFP) animal and labeled with DAPI. (D) Confocal scan of a transverse section across the dissected thyroid gland from Tg(tg:nls-EGFP) animal at 3 mpf. Sections were stained with DAPI to visualize cells surrounding thyroid follicles. (E) Boxplot depicting the proportion of thyrocytes present in transverse sections obtained from three Tg(tg:nls-EGFP) animals at 3 mpf. Each dot represents a transverse section. The Tukey boxplot marks the 25th percentile, median and 75th percentile with whiskers extending from smallest to largest values. (F , G) Representative FACS plot of single cells from Tg(tg:nls-mVenus-T2A-NTR) animals at 2 mpf (F) and 8 mpf (G). Calcein (Pacific Blue) labels live cells, while green fluorescence (FITC) labels thyrocytes. Percentage values represent proportion of calcein+ thyrocytes within total calcein+ cells.
Figure 2(A) A t-SNE plot displaying the 6249 single-cells profiled in the zebrafish thyroid gland atlas. The colors represent cell clusters denoting a specific cell-type. (B-D) Cluster #1 represents the thyrocytes that express tg, slc5a5 (NIS) and tpo. The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (red).
Figure 3(A) Heatmap depicting five genes specifically expressed in each one of the seven clusters of the zebrafish thyroid gland atlas. The color scale represents expression as a z-score ranging from -2 (purple) to 2 (yellow). NFE: Non-Follicular Epithelium.(B - G) t-SNE plots overlaid with the expression of a gene specific to each of the cluster. The endothelium cluster (cluster #2) is a mix of blood vessels and lymphatic vessels while the immune cell cluster (cluster #4) is a mix of macrophages, neutrophils and lymphocytes The color scale represents the normalized expression counts for each gene ranging from lowest (grey) to highest (blue).
Figure 4(A-D) Images show immunofluorescence labeling of thyroid gland from adult zebrafish. Transverse sections were utilized for imaging. The organ was isolated from tissue-specific transgenic lines to allow marking of a particular cell-type adjacent to the thyroid follicle. Blood vessels were marked using Tg(kdrl:EGFP) (A), macrophages using Tg(mpeg1.1:mCherry) (B) and stroma using Tg(col1a2:mCherry) (C). Thyrocytes were labeled with pax2amKO2 expression in (A) and Tg(tg:nls-EGFP) expression in (B - C). (D) NFE was labeled using antibody against TP63 in sections of the thyroid gland isolated from Tg(tg:nls-EGFP) animals. Gills are marked based on their morphological appearance. DAPI labels nuclei. Scale bars: 10 µm (A - B), 50 µm (C - D).
Figure 5(C) A dotplot depicting examples of paracrine and autocrine signaling in the thyroid gland. The size of the dots represent percentage of cells expressing the gene in a particular cluster, while the color scale represents average expression of the gene in the cluster after scaling.