Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: PTH infusion increased SAA3 expression and decreased femoral BMD in WT mice. WT and SAA3 KO male mice (3.5 mo old) were infused with vehicle (VEH) or PTH (40 µg/kg/d) for 12 d. (a) Saa3 mRNA expression in tibiae of WT mice. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by quantitative real-time PCR (qPCR) and data were reported as RQ values. (b) SAA3 protein in serum of WT and SAA3 KO mice by ELISA. Und, undetectable. (c) Change in in vivo femoral BMD calculated as: (BMD at end of infusion − BMD at start of infusion)/BMD at start of infusion. (d) Saa1, Saa2/Saa1, and (e) Ptgs2 (COX2) mRNA in WT and KO tibiae. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by qPCR and data were reported as RQ values. Bars are means ± SEM for n = 6 mice per genotype and treatment group. For (a), **P < 0.01, determined by two-tailed, unpaired t test. For (c) and (e), **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vivo, Two Tailed Test

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: PTH infusion increased trabecular bone formation parameters only in SAA3 KO mice but increased resorption parameters similarly in both WT and SAA3 KO mice. WT and SAA3 KO male mice were infused with vehicle (VEH) or PTH. (a) Static histomorphometry of trabecular bone in distal femurs for Ob.S/BS, Oc.S/BS, and eroded surface per bone surface (ES/BS). (b) Representative TRAP-stained (red) and counterstained hematoxylin ×200 original magnification (scale bars, 100 µm) microscopic images of trabecular bone in distal femurs. Plump cuboidal osteoblasts around trabeculae are marked with yellow arrows. (c) Dynamic histomorphometry of trabecular bone in distal femurs for MS/BS, MAR, and BFR/BS. (c) Representative calcein-labeled (green) and demeclocycline-labeled (orange-brown) ×400 original magnification (scale bars, 25 µm) microscopic images of trabeculae. (e and f) Measurement of (e) serum bone formation markers, PINP, and BGLAP (osteocalcin) and (f) serum bone resorption markers, CTX and TRAcP5b, measured by ELISA. (g and h) mRNA expression of Runx2, Bglap (osteocalcin), Tnfsf11 (RANKL), and Tnfrsf11b (OPG) in the tibiae measured by quantitative real-time PCR and reported as RQ values. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. Bars are means ± SEM for n = 6 mice per genotype and treatment group. *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Staining, Labeling, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: Continuous PTH increased osteoblast differentiation in hBMSCs only when COX2 or RANKL activity was blocked. hBMSCs were cultured with vehicle (VEH) or PTH (10 nM), with or without NS398 (100 nM), a selective inhibitor of COX2, or OPG (100 ng/mL), which blocks RANKL binding to its receptor. (a) Measurement of accumulated medium PGE2 by ELISA. (b) Markers of osteoblast differentiation, ALPL (alkaline phosphatase) and BGLAP (osteocalcin), and alizarin red staining for mineralization at day 21. (c) TNFSF11 (RANKL) mRNA at day 7 and TRAP staining at day 8. Scale bar, 100 µm. (d) BGLAP mRNA and alizarin red staining at day 21. (e) RUNX2 mRNA at day 7 and IGF1 and BMP2 mRNA at day 21. (f) WNT10B and DKK1 mRNA at day 7. mRNA was measured by quantitative real-time PCR and data are reported as RQ values. Bars are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (c), **P < 0.01, determined by two-tailed unpaired t test. For (d)–(f), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. Und, undetectable.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Activity Assay, Cell Culture, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: NS398 blocked the RANKL induction of SAA1 and SAA2 expression in hBMMs but did not affect osteoclast-like cell numbers, and SAA1 and SAA2 inhibited PTH-stimulated osteoblastic differentiation in hBMSCs. (a–c) hBMMs were treated with M-CSF (30 ng/mL) plus vehicle (VEH) or M-CSF plus RANKL (30 ng/mL each) with/without NS398 (100 nM). (a) SAA1 and SAA2 protein measurement in the culture medium of hBMMs measured by ELISA. (b) SAA1 and SAA2 mRNA at day 3. (c) TRAP-stained microscopic images at ×100 original magnification (scale bars, 200 µm) and TRAP+ MNC counts per well. (d) ALPL (alkaline phosphatase) and BGLAP (osteocalcin) mRNA expression in hBMSCs at day 21 treated with VEH or PTH in presence of OPG (100 ng/mL) to prevent osteoclastogenesis with/without rhSAA1 (10 µg/mL) or rhSAA2 (10 to 50 ng/mL). Data are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, significantly different from VEH treated at same time point, determined by two-tailed unpaired t test. For (c), **P < 0.01, significantly different from day 4, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (b) and (d), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer subtype

doi: 10.1101/2024.06.25.600311

Figure Lengend Snippet: A. Schematic overview of workflow for CAF isolation and TGFβ1 pathway-targeted qPCR array. B. RNA expression of TGFβ1 target genes in two primary CRC CAFs (CAF1 and CAF2) and one LM-CRC CAF. qPCR values have been log transformed. C. Numerical values depicting the seven TGFβ1 target genes that showed consistent upregulation. D. Fold change RNA expression of these seven TGFβ1 target genes (yellow symbols). E. IL6 RNA expression in primary CRC CAFs (CAF4 and CAF5) and the CCD-18Co colon fibroblast line after stimulation with TGFβ1 or medium control. N=3 CCD-18Co, CAF4; N=2 CAF5 independent biological experiments. ** p≤0.01 determined by paired T test. F. Protein expression of IL-6 in tissue lysates of normal colon and primary CRC (left panel, N=50) or normal liver and LM-CRC (right panel, N=50) as determined by ELISA. **** p≤0.0001, * p≤0.05 determined by unpaired T test. G. IL6 RNA expression in unstimulated normal colon fibroblasts (N=8) or primary CRC CAFs (N=10). H. RNA expression of IL6 in the original CMS classified cohort (CMS1: N=457; CMS2: N=1183; CMS3: N=409; CMS4: N=773). **** p≤0.0001 determined by one-way ANOVA with correction for multiple testing (Dunnett’s test).

Article Snippet: SAA1 concentrations in patient sera were measured using a commercially available kit (Human SAA1 DuoSet ELISA, R&D Systems) and a decrease of ≥0.3 OD450 value between post and pre-surgery sera was considered significant.

Techniques: Isolation, RNA Expression, Transformation Assay, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer subtype

doi: 10.1101/2024.06.25.600311

Figure Lengend Snippet: A. RNA expression of IL6 in CAFs derived either from the primary tumor (CRC CAF, N=18) or liver metastasis (LM-CRC CAF, N=6). *** p≤0.001 determined by unpaired T test. B. RNA-seq data from GSE46824 dataset showing IL6 RNA expression in an independent cohort of primary CRC CAFs (N=14) and LM-CRC CAFs (N=11). *** p≤0.001 determined by unpaired T test. C. IL-6 protein expression in unstimulated primary CRC CAF (N=8) and LM-CRC CAF (N=6) supernatant determined by ELISA. ** p≤0.01 determined by unpaired T test. D, E. IL-6 protein expression in TGFβ1-stimulated (5 ng/ml) primary CRC CAFs (N=8) ( D ) and LM-CRC CAFs (N=6) ( E ). * p≤0.05 determined by unpaired T test. F. Fold change IL-6 protein expression in supernatant from TGFβ1-stimulated vs. unstimulated primary CRC CAFs (N=8) and LM-CRC CAFs (N=6). ns, p>0.05 determined by unpaired T test. G. Percentage decrease in IL-6 protein expression in supernatant in primary CRC (N=5) and LM-CRC (N=5) CAFs after inhibition with SB431532 (Alk5 inhibitor). ns, p>0.05 determined by unpaired T test. H. RNA-seq data from GSE46824 dataset (2B) showing TGFβ1 RNA expression in an independent cohort of primary CRC CAFs (N=14) and LM-CRC CAFs (N=11). * p≤0.05 determined by unpaired T test. I. TGFβ1 protein expression in unstimulated primary CRC CAF (N=5) and LM-CRC CAF (N=6) supernatant determined by ELISA. * p≤0.05 determined by unpaired T test.

Article Snippet: SAA1 concentrations in patient sera were measured using a commercially available kit (Human SAA1 DuoSet ELISA, R&D Systems) and a decrease of ≥0.3 OD450 value between post and pre-surgery sera was considered significant.

Techniques: RNA Expression, Derivative Assay, RNA Sequencing, Expressing, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: bioRxiv

Article Title: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer subtype

doi: 10.1101/2024.06.25.600311

Figure Lengend Snippet: A-C. RNA expression of the three TGFβ isoforms in the original CMS cohort (CMS1: N=457; CMS2: N=1183; CMS3: N=409; CMS4: N=773). **** p≤0.0001 determined by one-way ANOVA with correction for multiple testing (Dunnett’s test). D. Protein expression of TGFβ isoforms in the supernatant of primary CRC and LM-CRC CAFs 48 hours after start of serum starvation as determined by ELISA. E, F. Expression of IL-6 in supernatant of primary CRC CAFs (N=7) ( E ) or LM-CRC CAFs (N=6) ( F ) 48 hours after start stimulation with either TGFβ2 or TGFβ3. * p≤0.05 determined by paired T test.

Article Snippet: SAA1 concentrations in patient sera were measured using a commercially available kit (Human SAA1 DuoSet ELISA, R&D Systems) and a decrease of ≥0.3 OD450 value between post and pre-surgery sera was considered significant.

Techniques: RNA Expression, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer subtype

doi: 10.1101/2024.06.25.600311

Figure Lengend Snippet: A. Schematic overview of ‘CAF priming of Huh-7 hepatocytes’, referring to Huh-7 cells are exposed to TGFβ1-stimulated CRC CAF CM B. Western blot for pSTAT3 and total STAT3 in wildtype Huh-7 cells that were stimulated with CAF CM or TGFβ1 CAF CM from 3 different CRC CAF lines. C. RNA expression of SAA1 and CXCL5 in wildtype Huh-7 cells stimulated with DMEM 0% FCS (Control), IL-6 (50 ng/ml), CAF CM or TGFβ1 CAF CM. N=2 or N=3 for the Control group, due to the very low SAA1 expression; for the other conditions, N=3.*** p≤0.001, ** p≤0.01, * p≤0.05 determined by one-way ANOVA with correction for multiple testing (Dunnett’s test). D, E RNA expression of SAA1 and CXCL5 in wildtype Huh-7 cells after CAF priming with blockade of specific components of the IL-6 signaling pathway. Siltuximab (anti-IL-6, 2 µg/mL), tocilizumab (anti-IL6R, 8 µg/mL), α-gp130 (anti-gp130, 8 µg/mL), tofacitinib (anti-JAK1/3, 5 µM), or human IgG isotype control (Bio X Cell; 2 µg/mL) were added to Huh-7 cells along with TGFβ1-stimulated CAF CM for 15 minutes. Subsequently, these different stimulations were applied to Huh-7 cells for 10 minutes. N=1 for all different conditions. **** p≤0.0001, *** p≤0.001 determined by one-way ANOVA with correction for multiple testing (Dunnett’s test).

Article Snippet: SAA1 concentrations in patient sera were measured using a commercially available kit (Human SAA1 DuoSet ELISA, R&D Systems) and a decrease of ≥0.3 OD450 value between post and pre-surgery sera was considered significant.

Techniques: Western Blot, RNA Expression, Control, Expressing

Journal: bioRxiv

Article Title: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer subtype

doi: 10.1101/2024.06.25.600311

Figure Lengend Snippet: A. Western blot for pSTAT3 and total STAT3 in CAF CM-primed Huh-7 cells in which gp-130 signaling is blocked using α-gp130 antibody. α-gp130, anti-gp130. β-actin is used as a loading control. B. Western blot for pSTAT3 and total STAT3 in vector control, gp130 KO , or gp130 KO rescued Huh-7 cells (gp130 KO+Rescue ) after stimulation with DMEM 0% (Control) or TGFβ1 CAF CM. C. RNA expression of SAA1 in gp130 KO and gp130 KO+Rescue Huh-7 cells after CAF priming (N=3 independent biological experiments). ** p≤0.01 determined by unpaired T test. D. IHC of pSTAT3 in Huh-7 cells harboring a vector control, gp130 KO or gp130 KO+Rescue after stimulation with DMEM 0% or after CAF priming. Scale bar, 50µm.

Article Snippet: SAA1 concentrations in patient sera were measured using a commercially available kit (Human SAA1 DuoSet ELISA, R&D Systems) and a decrease of ≥0.3 OD450 value between post and pre-surgery sera was considered significant.

Techniques: Western Blot, Control, Plasmid Preparation, RNA Expression

Journal: bioRxiv

Article Title: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer subtype

doi: 10.1101/2024.06.25.600311

Figure Lengend Snippet: A. Schematic overview of the KPN GEMM organoid experiment. B. Gene Set Enrichment analysis showing the Enrichment Score (ES) for the IL6-JAK-STAT signaling hallmark in KPN tumor tissue (N=3) and wildtype normal colon (N=3). C . Normalized counts of IL6, IL11 and LIF in KPN tumor tissue (KPN) and normal colon tissue (WT). **** p≤0.0001, ** p≤0.01 determined by unpaired T test. D. Quantification of automated scoring of Ly6G staining in pre-metastatic livers of KPN transplanted mice (N=4) and WT livers (N=5). E. Representative IHC of pSTAT3 and Ly6G in pre-metastatic and metastatic livers in the KPN GEMM. Scale bar, 50µm. F. Waterfall plot of difference in serum SAA1 OD450 value before and after surgery in 16 patients with primary CRC without liver metastasis.

Article Snippet: SAA1 concentrations in patient sera were measured using a commercially available kit (Human SAA1 DuoSet ELISA, R&D Systems) and a decrease of ≥0.3 OD450 value between post and pre-surgery sera was considered significant.

Techniques: Staining

Journal: bioRxiv

Article Title: TGFβ signaling in cancer-associated fibroblasts drives a hepatic gp130-dependent pro-metastatic inflammatory program in CMS4 colorectal cancer subtype

doi: 10.1101/2024.06.25.600311

Figure Lengend Snippet: Schematic overview of molecular mechanisms driving hepatic metastasis in CMS4 CRC.TGFβ stimulation on primary CRC CAFs induces the secretion of IL-6 family members, notably IL-6 and IL-11. Subsequently, these CAF-derived IL-6 and IL-11 activate a gp130-dependent STAT3 signaling pathway in hepatocytes. This, in turn, leads to the production of neutrophil chemoattractants such as SAA1 and CXCL5, facilitating the migration of pro-metastatic neutrophils toward the liver. Ultimately, this signaling cascade shapes a pre-metastatic hepatic milieu favorable for the seeding of primary CRC tumors.

Article Snippet: SAA1 concentrations in patient sera were measured using a commercially available kit (Human SAA1 DuoSet ELISA, R&D Systems) and a decrease of ≥0.3 OD450 value between post and pre-surgery sera was considered significant.

Techniques: Derivative Assay, Migration

Journal: PLoS Pathogens

Article Title: Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication

doi: 10.1371/journal.ppat.1006048

Figure Lengend Snippet: ( A ) Longitudinal proportion (%) of plasma genera detected in contemporaneous stool. Each line corresponds to a single animal. Bonferroni-corrected one-way ANOVA was used to calculate statistical significance. ( B ) Longitudinal plasma levels of IFABP, a marker of enterocyte loss and generalized damage to the intestinal epithelium. The host response to microbial translocation was measured using plasma levels of ( C ) EndoCAb, and ( D ) sCD14. By linear regression analysis, plasma levels of sCD14 at ( E ) 8 DPI, and ( F ) 21 DPI correlated positively with chronic-phase set-point viral loads. Acute inflammation was measured using plasma levels of ( G ) MCP-1, and ( H ) SAA1. For B, C, D, G, and H, differences between 0–8 DPI were evaluated for statistical significance by two-tailed Wilcoxon signed rank testing.

Article Snippet: Plasma SAA1 and IFABP were quantified using a commercially available Monkey SAA1 ELISA kit (MyBioSource, San Diego, CA) and Monkey IFABP/FABP2 ELISA kit (MyBioSource, San Diego, CA) with 1:1000 and 1:2 sample dilution, respectively.

Techniques: Clinical Proteomics, Marker, Translocation Assay, Two Tailed Test