Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: (A) 2303 natural compound libraries were tested for their ability to inhibit the interaction between recombinant human IL-1β and IL1 Receptor 1. Microtiter 96-well plates were coated with hIL-1β (100 ng/well) overnight and the blocking buffer without any single compound was used as a positive control. Diluted single compounds (20 μM) were added to each well and incubated for 2 h. After washing, recombinant human IL-1R1 (125 ng/ml) was added and incubated for 2 h. Subsequently, HRP-conjugated Anti-Human IgG Fc (1:2000) was added and incubated for 1 h. An OD450 was obtained following the TMB reaction. Data indicate mean ± SD (n = 3). (B) Dose-dependency test of selected natural compound for blocking the interaction between recombinant human IL-1β and IL-1R1. Every step was identical to primary screening except for concentrations of selected natural compound (10, 40, or 160 μM) and washing buffer (PBS containing 0.05% Tween-20 and 0.01% Triton X-100). * p <0.05 compared to the control group of IL-1β and IL1 Receptor 1 interaction without any natural compounds. (C) IL-1β-dependent HEK-Blue IL-1β cells (5×10 4 cell/well) were seeded onto a 96-well plate and treated with pre-incubation (20 min) of human IL-1β (10 ng/ml) with various concentrations of TA (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 μM). After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ and the optical ensity (OD) at 620 nm. Cell viability was measured by analyzing the OD at 450 nm using D-Plus CCK. The IC 50 value of tannic acid was determined using the GraphPadPrism 10 software. Data indicate mean ± SD (n = 3). (D) Surface plasmon resonance (SPR) assay was used to analyze the direct binding of tannic acid to human IL-1β. Human IL-1β protein (50 μg/ml) was immobilized on a CM5 sensor chip and various concentrations of TA (1.56, 3.125, 6.25, 12.5, 25, 37.5, 50, 62.5, 75, 87.5, or 100 μΜ) were injected into the flow system with a flow rate 20 μl/min for 300 s and allowed to dissociate for 600 s. The K D values of the tannic acid against human IL-1β were obtained using the T200 BIA evaluation software.

Article Snippet: ELISA-based binding assays were performed for IL-1β-blocking candidates to block the interaction between IL-1β and IL-1R1 using 2303 natural compound libraries (TargetMol L-6000, Wellesley Hills, MA, USA; Selleckhem L-1400, Matsonford Road Radnor, PA, USA).

Techniques: Recombinant, Blocking Assay, Positive Control, Incubation, Control, Activity Assay, Software, SPR Assay, Binding Assay, Injection

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: Human articular chondrocytes from OA patients were seeded onto 12-well plates (2×10 5 cells/well) and serum-starved cells were co-treated with various concentrations of TA (0.5, 1, or 2 μM) or IL-1R1 (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. (A) The mRNA expression levels of iNOS , COX-2 , IL-6 , and TNF were measured using qRT-PCR. The relative quantity of each gene expression was normalized to the relative quantity of human GADPH. (B) Griess reaction was used to measure the NO levels in the culture supernatants and PGE2, IL-6, and TNF levels in the culture supernatants were evaluated using ELISA. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

Article Snippet: ELISA-based binding assays were performed for IL-1β-blocking candidates to block the interaction between IL-1β and IL-1R1 using 2303 natural compound libraries (TargetMol L-6000, Wellesley Hills, MA, USA; Selleckhem L-1400, Matsonford Road Radnor, PA, USA).

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

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: Human articular chondrocytes from OA patients were seeded onto 6-well plates (3×10 5 cells/well) and serum-starved cells were co-treated with various concentrations of TA (2 μM), IL-1R1 (1 μg/ml), or anti-IL-1 neutralizing antibody (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. mRNA and protein expressions of MMPs, ADAMTSs, collagen type II, and aggrecan were detected by qRT-PCR (A) and Western blot analysis (B). The relative quantity of each gene expression was normalized to the relative quantity of human GADPH and β-actin was used as a loading control. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

Article Snippet: ELISA-based binding assays were performed for IL-1β-blocking candidates to block the interaction between IL-1β and IL-1R1 using 2303 natural compound libraries (TargetMol L-6000, Wellesley Hills, MA, USA; Selleckhem L-1400, Matsonford Road Radnor, PA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Gene Expression, Control

Journal: NPJ Breast Cancer

Article Title: IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer

doi: 10.1038/s41523-021-00305-w

Figure Lengend Snippet: a Quantification (photons/sec (p/s)) of primary tumour growth in IL1R1 fl/fl ( n = 8 primary tumours from n = 4 mice) and IL1R1 −/− ( n = 8 primary tumours from n = 4 mice) mice up to 12 days of post-orthotopic injection of E0771-luc2-V5-GFP cells and b correspondent micrographs. c Quantification (p/s) of primary tumour growth in IL-1B fl/fl ( n = 7) and IL-1B −/− ( n = 6) mice up to 26 days of post-orthotopic injection of E0771-luc2- GFP cells and d correspondent micrographs. IL-1B fl/fl : n = 13 primary tumours from n = 7 mice (day 7 and 14); n = 12 primary tumours from n = 6 mice (day 20 and 26). IL-1B −/− : n = 12 primary tumours from n = 6 mice (day 7); n = 10 primary tumours from n = 6 mice (day 14, 20, and 26). 2.3-fold increase in primary tumour growth in IL-1B −/− mice (7.6 × 10 8 p/s) compared to IL-1B fl/fl mice (3.2 × 10 8 p/s) ( P = 0.01). Data are mean +/− SEM, Two-way ANOVA with Sidak’s post-hoc test.

Article Snippet: The following plasmids were used for overexpression studies: IL-1B (MR226719L4, Origene), IL1R1 (MR227508L4, Origene), GFP (17448 pLenti CMV GFP Puro (658-5), Addgene), Luciferase (21474 pLenti CMV V5-LUC Blast (w567-1), Addgene).

Techniques: Injection

Journal: NPJ Breast Cancer

Article Title: IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer

doi: 10.1038/s41523-021-00305-w

Figure Lengend Snippet: a , b Images and quantification of primary tumour development in IL-1B fl/fl ( n = 8 primary tumours from n = 4 mice) and IL-1B −/− ( n = 10 primary tumours from n = 5 mice) mice after intra-ductal administration of E0771 Luc2 V5 IL-1B-GFP cells. Data are mean +/− SEM, Two-way ANOVA with Sidak’s post-hoc test. c , d Quantification of F4/80 + macrophages ( c ) and CD163 + macrophages ( d ) in tumour core and periphery in IL-1B fl/fl and IL-1B −/− mice. Data are mean ± SEM, Two-way ANOVA with Sidak’s post hoc test. e , f Quantification of CD34 + blood vessels and MPO + neutrophils in IL-1B fl/fl and IL-1B −/− mice. Data are shown as mean +/− SEM, Two-tailed unpaired t -test. g , h Images and quantification of primary tumour development in IL1R1 fl/fl ( n = 18 primary tumours from n = 9 mice) and IL1R1 −/− ( n = 14 primary tumours from n = 7 mice) mice after intra-ductal administration of E0771 Luc2 V5 IL-1B-GFP. Normalised data are shown as mean +/− SEM, Two-tailed unpaired t -test.

Article Snippet: The following plasmids were used for overexpression studies: IL-1B (MR226719L4, Origene), IL1R1 (MR227508L4, Origene), GFP (17448 pLenti CMV GFP Puro (658-5), Addgene), Luciferase (21474 pLenti CMV V5-LUC Blast (w567-1), Addgene).

Techniques: Two Tailed Test

Journal: NPJ Breast Cancer

Article Title: IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer

doi: 10.1038/s41523-021-00305-w

Figure Lengend Snippet: a , b Tumour proliferation after injection of E0771 luc2 V5 IL1R1-GFP cells in IL1R1 fl/fl ( n = 8 primary tumours from n = 4 mice) and IL1R1 −/− ( n = 6 primary tumours from n = 3 mice) mice. Data are mean +/− SEM, Two-way ANOVA with Sidak’s multiple comparisons test. c In vitro relative tumour growth of E0771 luc2 V5 GFP, IL-1B GFP or IL1R1 GFP cells upon stimulation with 40 pg/ml mouse recombinant IL-1B. Data are mean (+/− SEM), Two-way ANOVA with Sidak’s multiple comparison test ( n = 6 technical repeats from two biological replicates). d Transwell cell migration of IL-1B overexpressing ( P = 0.0009) and IL1R1 overexpressing ( P < 0.0001) E0771 luc2 V5 cancer cells compared to control GFP-expressing cells (no treatment with exogenous IL-1B). Comparison of cell migration in vitro between E0771 luc2 V5 IL-1B GFP and IL1R1 GFP tumour cells ( P = 0.0018). Data are mean (+/− SEM) cell number from 4 fields of view derived from three biological experiments ( n = 12 technical repeats). Two-tailed unpaired t -test with Welch’s correction. e Representative images of haematoxylin-stained control, IL-1B and IL-1R1 overexpressing cells migrated through the membrane. Scale bar = 200 µm.

Article Snippet: The following plasmids were used for overexpression studies: IL-1B (MR226719L4, Origene), IL1R1 (MR227508L4, Origene), GFP (17448 pLenti CMV GFP Puro (658-5), Addgene), Luciferase (21474 pLenti CMV V5-LUC Blast (w567-1), Addgene).

Techniques: Injection, In Vitro, Recombinant, Migration, Expressing, Derivative Assay, Two Tailed Test, Staining

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Specificity of the anti-IL-1R1 antibody and distribution of IL-1R1 immunoreactivity in the spinal dorsal horn. a Adsorption of anti-IL-1R1 antibody to recombinant IL-1R1 peptide completely abolished the immunostaining. b Western blot analysis reinforces the specificity of the anti-IL-1R1 antibody. The single immunoreactive band indicates that the antibody detects a protein with a molecular mass of ~80 kDa that corresponds to the molecular weight of IL-1R1. c , d Micrographs showing immunoreactivity for IL-1R1 in control ( c ) and CFA-injected rats ( d ) 3 days after CFA-injection. Bars 100 μm

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Adsorption, Recombinant, Immunostaining, Western Blot, Molecular Weight, Injection

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: CFA-evoked inflammation of the hindpaw initiates an overproduction of IL-1R1 protein in the spinal dorsal horn of rats. a Representative immune-blots showing immunoreactive bands for IL-1R1 and β-tubulin (loading control) in Western blots of tissue samples obtained from the L3–L5 lumbar segments of the spinal dorsal horn of the control and CFA-injected animals at post-injection day 3. b Histogram showing the optical densities of IL-1R1 immunostained bands (see on insert a ) calculated in proportion to the optical densities of β-tubulin (loading control) immunostained bands (see on insert a ). IL-1R1 protein level was found to be significantly higher than the control value ( p = 0.029). Data are shown as mean ± SEM

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Western Blot, Injection

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Mechanical withdrawal threshold and thermal withdrawal latency of wild type and IL-1R1 knockout mice during the course of CFA-induced inflammation of the hind paw. a The histogram shows the mechanical withdrawal threshold (MWT) of wild type (BL6) and IL-1R1 knockout (IL-1R1 KO) mice receiving different treatments in the three experimental groups: group (1) complete Freund-adjuvant (CFA) injection (BL6 CFA, IL-1R1 KO CFA); group (2) physiological saline injection (BL6 sham, IL-1R1 KO sham); and group (3) without any treatment (BL6 control, IL-1R1 KO control). Measurements were made only on the right hind paw which received the physiological saline or CFA injections. b The histogram shows the thermal withdrawal latency (TWL) of wild type (BL6) and IL-1R1 knockout (IL-1R1 KO) mice receiving different treatments in the three experimental groups: group (1) complet Freund-adjuvant (CFA) injection (BL6 CFA, IL-1R1 KO CFA); group (2) physiological salt solution injection (BL6 sham, IL-1R1 KO sham); and group (3) without any treatment (BL6 control, IL-1R1 KO control). Measurements were made only on the right hind paw which received the physiological saline or CFA injections. Data are shown as mean ± SEM

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Knock-Out, Injection

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Localization of IL-1R1 on neurons and glial cells in the superficial spinal dorsal horn of rats. Micrographs of single 1-μmthick laser scanning confocal optical sections illustrating the co-localization between immunolabeling for IL-1R1 ( red ; a – d , e , h , k , n , q ) and immunoreactivity for markers that are specific for axon terminals of peptidergic (CGRP, green ; a ) and non-peptidergic (IB4 binding, green ; b ) primary afferents, axon terminals of excitatory (VGLUT2, green ; c ) and inhibitory (VGAT, green ; d ) intrinsic neurons, astrocytes (GFAP, green ; o ) and microglial cells (CD11b, green ; r ), and postsynaptic membranes of excitarory (PSD95, green ; i ) and inhibitory (gephyrin, green ; l ) synapses in the superficial spinal dorsal horn. Mixed colors ( yellow ; marked by white arrowheads ) on the superimposed images ( j , m , p , s ) indicate double-labeled structures . The absence of yellow color on a – d indicates a lack of IL-1R1 expression on axon terminals of various origin. IL-1R1 immunoreactive spots appear in two different localization on the micrographs showing immunostaining also for KCC2 ( green , f , g ): (1) They can be aligned along the lines defined by the KCC2 immunostaining (cell membrane localization; white arrowheads on f and g ). (2) They can also be located in areas surrounded by the KCC2-immunostained cell membranes (cytoplasmic localization, yellow arrowhead on g . Bars 2 μm ( a – d ), 5 μm ( n – s ), and 10 μm ( e – m )

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Immunolabeling, Binding Assay, Labeling, Expressing, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Histogram showing the CFA-evoked inflammation induced changes in the degree of co-localization between immunoreactivity for IL-1R1 and selected neuronal and glial markers in the superficial spinal dorsal horn of rats. Columns indicate the percentages of profiles immunoreactive for IL-1R1 that were found to be labeled also for the selected markers, and the ones that were aligned along KCC2 immunoreactive membranes (localization on the somatodendritic membrane of neurons) or were located within areas surrounded by KCC2 immunoreactive membranes (localization within the cytoplasm of the somatodendritic compartment of neurons). White columns show data obtained from control animals, whereas black columns represent values found in CFA-injected animals 3 days after CFA injection into the right hind paw. Asterisk indicate that CFA-evoked inflammation significantly increased the number of spots immunoreactive for IL-1R1 on the somato-denditic membrane of neurons ( p = 0.000001). Data are shown as mean ± SEM

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Labeling, Injection

Journal: Cancer discovery

Article Title: IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.

doi: 10.1158/2159-8290.CD-18-0710

Figure Lengend Snippet: A. qPCR analysis of Il1a, Il1b, Tnf, Il1r1, Tnfrsf1a, and epithelial (Epcam and Cdh1) and fibroblast (Pdgfra, Pdpn and Col1a1) markers in EpCAM+ (epithelial cells) relative to PDPN+ (CAFs) cells sorted from KPC tumors. Results show mean ± SEM (standard error of the mean) of 6 biological replicates. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. B. Representative flow cytometric analysis of IL-1R1 in EpCAM+ (epithelial cells) and PDPN+ (CAFs) cells in KPC tumors (n=3). Percentages shown were calculated from the parental gate. C. Violin plots showing single cell RNA-sequencing analysis of Il1a, Il1b, Il1r1, Epcam and Col1a1 of a representative KPC tumor (n=2) in CAFs (orange) and epithelial cells (green). D. ELISA of IL-1α from media of mouse 2D KPC cells (n=2), tumor (T) (n=8) and metastatic (M) (n=8) organoids, and controls that do not induce the iCAF phenotype (n=2 for each control). Results show mean ± SEM. E. Western blot analysis of the nuclear factor NF-κB p65 subunit following nuclear fractionation of quiescent PSCs (qP, PSCs cultured in Matrigel with control media, i.e. 5% FBS DMEM, for 4 days), iCAFs (iC, PSCs cultured in Matrigel with tumor organoid-conditioned media for 4 days) and myCAFs (myC, PSCs cultured in monolayer with 5% FBS DMEM). Loading controls, HSP90α (cytoplasmic fractions) and H3 (nuclear fractions). The same amount of protein lysate was loaded in each lane. F. Western blot analysis of total and phosphorylated p65 (p-p65) and of total IκBα in PSCs cultured in Matrigel in control media or tumor organoid-conditioned media (CM) in the presence or absence of 30 μM IKK-β inhibitor (IKK-βi) ML102B for 30 min. Loading control, ACTIN. G. qPCR analysis of iCAF markers (Il1a, Il6, Lif, Cxcl1 and Csf3) in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media for 1 h in the presence or absence of 30 μM ML102B. Results show mean ± SEM of 3 biological replicates. *P<0.05, **P<0.01, paired Student’s t test.

Article Snippet: Wild-type mouse IL-1R1 cDNA (MC219163, Origene) was PCR amplified with mutagenic primers to induce a G>T transversion, thereby converting codon 8 from GGG>GGT. qPCR analysis.

Techniques: RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fractionation, Cell Culture

Journal: Cancer discovery

Article Title: IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.

doi: 10.1158/2159-8290.CD-18-0710

Figure Lengend Snippet: A. qPCR analysis of iCAF (Il1a, Il6, Lif, Cxcl1 and Csf3) and myCAF (Acta2 and Ctgf) markers in PSCs cultured in Matrigel in control media in the presence or absence of 1 ng/mL mouse IL-1α for 4 days. Results show mean ± SEM of 2 biological replicates. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. B. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence of a neutralizing antibody targeting IL-1α or an IgG control for 4 days. Results show mean ± SEM of 6 biological replicates. *P<0.05, **P<0.01, paired Student’s t test. C. Proliferation curves of PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence of a neutralizing antibody targeting IL-1α or an IgG control. Results show mean ± SEM of 3 biological replicates. **P<0.01, ***P<0.001, unpaired Student’s t test calculated for the last time point. D. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in PSCs cultured in Matrigel in transwell with Rosa26-targeted controls or IL-1α knockout (KO) tumor organoids for 4 days. Results show mean ± SEM of 9 and 11 biological replicates, respectively. ***P<0.001, paired Student’s t test. E. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in Rosa26-targeted controls and IL-1R1 knockout PSCs cultured in Matrigel in transwell with tumor organoids for 4 days. Results show mean ± SEM of 7 biological replicates. *P<0.05, ***P<0.001, paired Student’s t test. F. Proliferation curves of Rosa26-targeted controls and IL-1α knockout tumor organoids. Results show mean ± SD (standard deviation) of 5 technical replicates. *P<0.05, **P<0.01, unpaired Student’s t test calculated for the last time point. G. Tumor volume analysis based on ultrasound measurements of orthotopically grafted organoids (OGOs) following ~3 weeks from transplantation of Rosa26-targeted controls and IL-1α knockout tumor organoids in nu/nu mice. Results show mean ± SEM of 14 (control OGOs), 7 (1C or 1D OGOs) and 8 (1E OGOs) biological replicates. **P<0.01, ***P<0.001, unpaired Student’s t test. H. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in CAFs sorted from OGOs derived from transplantation of Rosa26-targeted controls and IL-1α knockout tumor organoids in nu/nu mice. Results show mean ± SEM of 4 (organoids), 12 (control OGOs) and 19 (IL-1α knockout OGOs) biological replicates. Different symbols identify the 3 knockout lines. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. I. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2, Ctgf and Il1r1 in CAFs sorted from tumors derived by orthotopic transplantation of 3 tumor organoid lines in IL-1R1 knockout or C57BL/6J controls. Results show mean ± SEM of 9 biological replicates. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. J. Quantification of Ly6C- myCAF/Ly6C+ iCAF ratio in tumors derived by orthotopic transplantation of 2 tumor organoid lines in B6J or IL-1R1 knockout hosts, as assessed by flow cytometry. Results show mean ± SEM of 5 biological replicates. **P<0.01, unpaired Student’s t test.

Article Snippet: Wild-type mouse IL-1R1 cDNA (MC219163, Origene) was PCR amplified with mutagenic primers to induce a G>T transversion, thereby converting codon 8 from GGG>GGT. qPCR analysis.

Techniques: Cell Culture, Knock-Out, Standard Deviation, Transplantation Assay, Derivative Assay, Flow Cytometry

Journal: Cancer discovery

Article Title: IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.

doi: 10.1158/2159-8290.CD-18-0710

Figure Lengend Snippet: A. qPCR analysis of iCAF (Il1a, Il6, Lif, Cxcl1 and Csf3) and myCAF (Acta2 and Ctgf) markers in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence or absence of 500 nM JAK inhibitor (JAKi) AZD1480 for 4 days. Results show mean ± SEM of 5 biological replicates. *P<0.05, **P<0.01, paired Student’s t test. B. Proliferation curves of PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence or absence of 500 nM JAKi. Results show mean ± SEM of 3 biological replicates. ***P<0.001, unpaired Student’s t test calculated for the last time point. C. RNA-sequencing analysis of quiescent PSCs (n=3), iCAFs (n=3) and iCAFs treated with 500 nM JAKi for 4 days (n=3). Color scheme represents Z-score distribution. D. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence or absence of 500 nM JAKi for the last 24 h following 4 days in culture with conditioned media. Results show mean ± SEM of 5 biological replicates. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. E. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in Rosa26-targeted controls and STAT3 knockout PSCs cultured in Matrigel in transwell with tumor organoids for 4 days. Results show mean ± SEM of 5 biological replicates. *P<0.05, ***P<0.001, paired Student’s t test. F. Western blot analysis of IL-1R1 in myCAFs, quiescent PSCs and iCAFs. Loading control, ACTIN. G. qPCR analysis of Il1r1 in PSCs cultured in Matrigel with tumor organoid-conditioned media in the presence or absence of 500 nM JAKi (left) and in STAT3 knockout PSCs compared to controls (right). Results show mean ± SEM of 4 and 5 biological replicates, respectively. ***P<0.001, paired Student’s t test. H. Representative immunofluorescence co-stains of p-STAT3 (green) and αSMA (red) in KPC tumor sections (n=4). Counterstain, DAPI (blue). Arrows indicate examples of αSMA+ p-STAT3- myCAFs, arrow-heads indicate examples of αSMA- p-STAT3+ cells, asterisks indicate examples of αSMA+ p-STAT3+ cells. Scale bars, 100 μm. I. Quantification of αSMA+ p-STAT3- cells and αSMA+ p-STAT3+ cells in KPC tumor sections. Results show mean ± SEM of 4 biological replicates. ***P<0.001, unpaired Student’s t test.

Article Snippet: Wild-type mouse IL-1R1 cDNA (MC219163, Origene) was PCR amplified with mutagenic primers to induce a G>T transversion, thereby converting codon 8 from GGG>GGT. qPCR analysis.

Techniques: Cell Culture, RNA Sequencing Assay, Knock-Out, Western Blot, Immunofluorescence

Journal: Cancer discovery

Article Title: IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.

doi: 10.1158/2159-8290.CD-18-0710

Figure Lengend Snippet: A. Western blot analysis of SMAD2, p-SMAD2, p-SMAD3, SMAD3, CTGF and αSMA in myCAFs (myC), quiescent PSCs (qP) and iCAFs (iC). Loading control, HSP90α. B. Western blot analysis of the TGF-β signaling effector SMAD4 following nuclear fractionation of quiescent PSCs (qP), iCAFs (iC) and myCAFs (myC). Loading controls, HSP90α (cytoplasmic fractions) and H3 (nuclear fractions). The same amount of protein lysate was loaded in each lane. C. Violin plots showing single cell RNA-sequencing analysis of Ctgf and Col1a1 of a representative KPC tumor (n=2) in myCAFs (blue) and iCAFs (orange). D. Representative immunofluorescence co-stains of p-SMAD2 (green) and αSMA (red) in KPC tumor sections (n=5). Counterstain, DAPI (blue). Arrow-heads indicate examples of αSMA+ p-SMAD2+ cells. Scale bars, 100 μm. E. Quantification of p-SMAD2+ αSMA- cells and p-SMAD2+ αSMA+ cells in KPC tumor sections. Results show mean ± SEM of 5 biological replicates. **P<0.01, paired Student’s t test. F. qPCR analysis of iCAF (Il1a, Il6, Lif, Cxcl1 and Csf3) and myCAF (Acta2 and Ctgf) markers in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence or absence of 20 ng/mL mouse TGF-β for 4 days. Results show mean ± SEM of 6 biological replicates. **P<0.01, ***P<0.001, paired Student’s t test. G. Western blot analysis of p-JAK1, JAK1, p-JAK2, JAK2, p-STAT1, STAT1, p-STAT3, STAT3, p-SMAD2, CTGF and αSMA in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media (CM) in the presence or absence of 20 ng/mL mouse TGF-β for 4 days. Loading control, ACTIN. H. Proliferation curves of PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence or absence of 20 ng/mL mouse TGF-β. Results show mean ± SD of 5 technical replicates. *P<0.05, **P<0.01, ***P<0.001, unpaired Student’s t test calculated for the last time point. I. qPCR analysis of Il1r1 in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence or absence of 20 ng/mL mouse TGF-β for 4 days. ***P<0.001, paired Student’s t test. J. Western blot analysis of IL-1R1 in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence or absence of 20 ng/mL mouse TGF-β for 4 days. Loading control, ACTIN.

Article Snippet: Wild-type mouse IL-1R1 cDNA (MC219163, Origene) was PCR amplified with mutagenic primers to induce a G>T transversion, thereby converting codon 8 from GGG>GGT. qPCR analysis.

Techniques: Western Blot, Fractionation, RNA Sequencing Assay, Immunofluorescence, Cell Culture

Journal: Cancer discovery

Article Title: IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.

doi: 10.1158/2159-8290.CD-18-0710

Figure Lengend Snippet: A. Tumor volume analysis based on ultrasound measurements of vehicle- and JAK inhibitor (JAKi)- treated KPC tumors. Results show mean ± SEM of 8 and 7 tumors, respectively. *P<0.05, unpaired Student’s t test. B. Representative Hematoxylin and Eosin (H&E) stain of vehicle- and JAKi- treated KPC tumor sections (n=9 and 7, respectively). Scale bar, 200 μm. C. Representative Masson’s trichrome stain of vehicle- and JAKi- treated KPC tumor sections (n= 9 and 7, respectively). Scale bar, 200 μm. D. Quantification of Masson’s trichrome stain in vehicle- and JAKi- treated KPC tumors. Results show mean ± SEM of 9 and 7 biological replicates, respectiv12`ely. ***P<0.001, unpaired Student’s t test. E. Quantification of CAFs in vehicle- and JAKi- treated KPC tumors, as assessed by flow cytometry. Results show mean ± SEM of 4 biological replicates. *P<0.05, unpaired Student’s t test. F. Representative immunohistochemistry of αSMA stain of vehicle- and JAKi- treated KPC tumor sections (n=7). Scale bar, 200 μm. G. Quantification of αSMA stain in vehicle- and JAKi- treated KPC tumors. Results show mean ± SEM of 7 biological replicates. *P<0.05, unpaired Student’s t test. H. Representative flow cytometric analysis of iCAFs and myCAFs in EdU-treated KPC tumors (n=2). The values shown represent the EdU+ cells in each CAF population. I. Quantification of Ly6C- myCAF/Ly6C+ iCAF ratio in vehicle- and JAKi- treated KPC tumors, as assessed by flow cytometry. Results show mean ± SEM of 3 biological replicates. *P<0.05, unpaired Student’s t test. J. Model explaining the pathway antagonism that determines iCAF and myCAF formation in PDAC. (1) Tumor-secreted TGF-β activates TGF-β signaling in adjacent myCAFs, preventing induction of the iCAF phenotype by suppressing IL-1R1 expression. (2) Conversely, tumor-secreted IL-1 activates IL-1 signaling in CAFs that are located farther away from tumor glands. (3) In these CAFs, IL-1 signaling induces a cytokine cascade that leads to JAK/STAT signaling activation through NF-κB signaling and autocrine LIF. (4) The activated JAK/STAT pathway establishes a positive feedback loop by upregulating IL-1R1 expression.

Article Snippet: Wild-type mouse IL-1R1 cDNA (MC219163, Origene) was PCR amplified with mutagenic primers to induce a G>T transversion, thereby converting codon 8 from GGG>GGT. qPCR analysis.

Techniques: Staining, Flow Cytometry, Immunohistochemistry, Expressing, Activation Assay