Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A–B) CD36 protein and mRNA levels detected by flow cytometry or qRT-PCR on Swiss murine peritoneal macrophages firstly treated during 24 h with TNF-α (10 ng/mL), peptidoglycan (PGN) (1 µg/mL) or Plasmodium falciparum culture supernatant ( P.f. c s) and incubated during 20 supplementary hours with rosiglitazone (R) (5 µM) or IL13 (50 ng/mL) for protein quantification or 5 supplementary hours for mRNA detection. Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with control cells (untreated). (C) CD36 protein level detected by flow cytometry on macrophages pre-incubated with a TL2 blocking monoclonal antibody (Anti-TLR2 mAb) (10 µg/mL) or Etanercept, a TNF-α inhibitor (10 µg/mL), and stimulated with TNF-α (10 ng/mL), PGN (1 µg/mL) or P.f . cs. Data are from a representative experiment performed in triplicate ± SD. **p<0.01 compared with control cells (untreated). ## p<0.01 compared with the respective control (Etanercept treated cells). (D) Phagocytosis index of P. falciparum unopsonized erythrocytes by Swiss murine macrophages stimulated as described in (A). Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with control cells (untreated). (E) PPARγ protein and mRNA levels in Swiss macrophages were determined by qRT-PCR after treatment of cells with TNF-α, PGN or P.f. cs. Data are represented as a mean ± SD of three independent experiments for mRNA quantification. **p<0.01 compared with control cells (C).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Flow Cytometry, Quantitative RT-PCR, Incubation, Control, Blocking Assay

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A–B) CD36 protein and mRNA levels detected by flow cytometry or qRT-PCR on Swiss murine peritoneal macrophages firstly treated during 24 h with TNF-α (10 ng/mL), PGN (1 µg/mL) or P. falciparum culture ( P.f. c s) and then incubated during 20 h with sulforaphane (SFN) (10 µM) or diethylmaleate (DEM) (100 µM) for protein quantification or 5 supplementary hours for mRNA detection. Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (TNF-α treated cells); ¤¤ p<0.01 compared with the respective control (PGN treated cells); §§ p<0.01 compared with the respective control ( P.f . cs treated cells); δδ p<0.01compared with control SFN-treated cells; ¶¶ p<0.01compared to control DEM-treated cells. (C) Phagocytosis index of Pf PEs by Swiss murine macrophages stimulated as described in (A). Data are represented as a mean ± SD of three independent experiments. *p<0.05 and **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (TNF-α treated cells); $$ p<0.01 compared with the respective control (SFN treated cells); ££ p<0.01 compared with the respective control (TNF+SFN treated cells) (D) HO-1 mRNA level in Swiss peritoneal macrophages determined by qRT-PCR after treatment of cells during 24 h with TNF-α (10 ng/mL) and then during 5 h with rosiglitazone (R) (5 µM), IL13 (50 ng/mL) or SFN (10 µM). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (TNF-α treated cells).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Flow Cytometry, Quantitative RT-PCR, Incubation, Control

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A) PPARγ protein level on Swiss murine peritoneal macrophages and on RAW264.7 cells. The experiments were repeated three times. (B) CD36 protein level on murine RAW264.7 cells after treatment of cells with IL13 (50 ng/mL), rosiglitazone (R) (5 µM), sulforaphane (SFN) (10 µM) or diethylmaleate (DEM) (100 µM). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with control cells. (C) CD36 protein level detected by flow cytometry on Swiss murine peritoneal macrophages firstly treated during 1 h with the PPARγ antagonists GW9662 (5 µM) and T007 (2 µM) and then incubated during 20 h with IL13, SFN or DEM. Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (GW9662 treated cells); §§ p<0.01 compared with the respective control (T007 treated cells). (D) CD36 mRNA level on PPARγ +/+ and PPARγ −/− C57BL/6 murine peritoneal macrophages after treatment of cells with rosiglitazone, IL13, SFN or DEM. Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with the respective control (PPARγ +/+ ); ## p<0.01 compared with the respective control (PPARγ −/− ). (E) Phagocytosis index of P. falciparum unopsonized erythrocytes by murine PPARγ +/+ and PPARγ −/− C57BL/6 macrophages stimulated as described in (E). Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with the respective control (PPARγ +/+ ); # p<0.05 compared with the respective control (PPARγ −/− ).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Control, Flow Cytometry, Incubation

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: CD36 mRNA level on Swiss peritoneal macrophages treated during 24 h with TNF-α (10 ng/mL) (A) and on RAW264.7 cells (B) and transfected with siRNA targeting Nrf2 (siRNA Nrf2) or control siRNA (siRNA control) and stimulated with sulforaphane SFN (10 µM) or diethylmaleate (DEM) (100 µM). Data are represented as a mean ± SD of three independent experiments. *p<0.05 and **p<0.01 compared with the respective control (cells transfected with siRNA control), (C) CD36 mRNA level on Nrf2 +/+ and Nrf2 −/− C57BL/6 murine peritoneal macrophages after treatment during 24 h with TNF-α and then incubated during 5 h with SFN or DEM. Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times **p<0.01 compared with the respective control (Nrf2 +/+ ), ## p<0.01 compared with the respective control (Nrf2 +/+ cells treated with TNF-α). (D) Phagocytosis index of Pf PEs by murine Nrf2 +/+ and Nrf2 −/− C57BL/6 macrophages stimulated as described in (C). Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with the respective control (Nrf2 +/+ control cells); # p<0.05 compared with the respective control (Nrf2 +/+ cells treated with TNF-α).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Transfection, Control, Incubation

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A) CD36 protein level detected by flow cytometry on human-monocytes derived macrophages (hMDMs) firstly treated during 24 h with TNF-α (10 ng/mL) or PGN (1 µg/mL) and then incubated during 20 h with rosiglitazone (R) (5 µM), IL13 (50 ng/mL), SFN (10 µM) or DEM (100 µM). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); # p<0.05 compared with the respective control (TNF-α treated cells); ¤¤ p<0.01 compared with the respective control (PGN treated cells). (B) PPARγ protein and mRNA levels on hMDMs treated with TNF-α, LPS, P.f . cs and PGN. PPARγ western blot is representative of three independent experiments and qPCR data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated). (C) Phagocytosis index of P. falciparum unopsonized erythrocytes by hMDMs stimulated as described in (A). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (PGN treated cells); $$ p<0.01 compared with the respective control (SFN treated cells); ££ p<0.01 compared with the respective control (TNF+SFN treated cells).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Flow Cytometry, Derivative Assay, Incubation, Control, Western Blot

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: Swiss mice receiving PGN (200 µg/mouse) (subcutaneous route) two days before infection and d,L-sulforaphane (75 mg/kg) or rosiglitazone (3 mg/kg) (oral route) during five days postinfection were infected with 1×10 6 Plasmodium berghei parasites via intra-peritoneal injection. (A) and (C) Survival was assessed twice daily. **p<0.01 compared with untreated mice (control), ¤¤ p<0.01 compared with PGN-treated mice. (B) and (D) Parasitemia levels were assessed daily. **p<0.01 compared with untreated macrophages (control); ¤ p<0.05 and ¤¤ p<0.01 compared with PGN-treated cells. (E) and (F) CD36 expression on macrophages was measured the day of infection by flow cytometry on three independent mice. *p<0.05 compared with untreated macrophages (control); ¤ p<0.05 compared with PGN-treated cells. (G) Phagocytosis index of P. berghei unopsonized erythrocytes by Swiss murine macrophages incubated with FcR blocking antibodies (20 µg/mL) and CD36 blocking antibodies (10 µg/mL). Data are presented as mean ± SD of one experiment performed in triplicate. **p<0.01 compared to uRBC+α−FcR, $ p<0.05 compared with iRBC+α−CD36, £ p<0.05 compared with iRBC+α−FcR. (H) Phagocytosis index of P. berghei unopsonized erythrocytes by 3 days-infected macrophages from Swiss mice treated with PGN and SFN as described above. ¤ p<0.05 compared with PGN-treated macrophages.

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Infection, Injection, Control, Expressing, Flow Cytometry, Incubation, Blocking Assay

Journal: Materials Today Bio

Article Title: A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces

doi: 10.1016/j.mtbio.2023.100727

Figure Lengend Snippet: Schematic illustration of PBPI 3 -based surface functionalisation inside porous scaffolds for MSC expansion: a) 3D printing of polymer scaffold; b) Plasma activation inside porous scaffolds for PBPI 3 surface activation; c) Reagent-free covalent immobilisation of FGF2 onto PBPI 3 -activated scaffolds for MSC expansion.

Article Snippet: Surface-bound FGF2 was detected with 1 μg/mL mouse monoclonal anti-FGF2 antibody (Abcam) for 1 h at RT and washed three times with PBS.

Techniques: Polymer, Clinical Proteomics, Activation Assay

Journal: Materials Today Bio

Article Title: A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces

doi: 10.1016/j.mtbio.2023.100727

Figure Lengend Snippet: a) Quantification of surface-bound bovine serum albumin (BSA) on untreated and PBPI 3 -treated scaffolds with and without SDS washing via a BCA assay. b) Fluorescent images of Cy5-conjugated antibody coated on untreated and PBPI 3 -treated scaffolds before and after Tween-20 washing. Scale bars are 400 μm or 3 mm as indicated. c) Antibody detection of surface-bound fibroblast growth factor 2 (FGF2) on untreated and PBPI 3 -treated scaffolds before and after Tween-20 washing. d) Antibody detection of surface-bound FGF2 on untreated and PBPI 3 -treated scaffolds after being coated with 0–640 ng/mL of FGF2. (*p < 0.05; ***p < 0.001; ****p < 0.0001).

Article Snippet: Surface-bound FGF2 was detected with 1 μg/mL mouse monoclonal anti-FGF2 antibody (Abcam) for 1 h at RT and washed three times with PBS.

Techniques: BIA-KA

Journal: Materials Today Bio

Article Title: A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces

doi: 10.1016/j.mtbio.2023.100727

Figure Lengend Snippet: MSC proliferation over 7 days on a) Untreated (UT) or b) PI 3 -treated 2D polystyrene films coated with 0–1000 ng/mL FGF2, or with 1000 ng/mL FGF2 supplemented in culture medium. Cell numbers were measured by DNA quantification at 4- and 7-days post-seeding. The asterisk (*) symbol denotes statistical comparison with the no-FGF2 control (UT 0 or PI 3 0); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The phi (Φ) symbol represents statistical comparison with FGF2 in solution conditions (UT sol 1000 or PI 3 sol 1000); Φ p < 0.05; Φ Φ p < 0.01; Φ Φ Φ p < 0.001; Φ Φ Φ Φ p < 0.0001. c) Representative images of crystal violet stained MSCs cultured for 7 days on untreated and PI 3 -treated films coated with 0–1000 ng/mL FGF2. Scale bar: 1 mm. d) Representative images of crystal violet stained MSCs cultured for 14 days on untreated and PBPI 3 -treated 3D scaffolds coated with 0–2000 ng/mL FGF2. Scale bar: 1 mm. e-f) Cell numbers determined from metabolic activity as measured with Alamar Blue assay. Cells were grown on e) untreated scaffolds or f) PBPI 3 - treated scaffolds, with or without FGF2 coating at 20, 200, and 500 ng/mL.

Article Snippet: Surface-bound FGF2 was detected with 1 μg/mL mouse monoclonal anti-FGF2 antibody (Abcam) for 1 h at RT and washed three times with PBS.

Techniques: Comparison, Control, Staining, Cell Culture, Activity Assay, Alamar Blue Assay

Journal: Materials Today Bio

Article Title: A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces

doi: 10.1016/j.mtbio.2023.100727

Figure Lengend Snippet: MSC proliferation and phenotype changes over 14 days on 3D scaffolds with or without PBPI 3 surface modification and FGF2 functionalisation. a) Cell abundance on untreated and PBPI 3 -treated scaffolds coated with 0, 10, or 20 ng/mL FGF2 at 3-, 7-, 10- and 14-days post-seeding (*p < 0.05). b-c) Fluorescent images of MSCs, stained for f-actin (red) on untreated and treated scaffolds with or without 20 ng/mL FGF2, at 1, 4, 7, 10, 12 and 14 days post-seeding. Fluorescent intensity, as a measure of cell abundance, is depicted by a LUT colour scale from black (low), red (medium), to yellow (high). Images were taken b) across the whole scaffold (scale bar: 4 mm). and c) with magnified snapshots provided (scale bar: 1 mm). d-f) Surface marker expression of MSCs expanded on tissue culture plastic (C), or on PBPI 3 -treated HiPS scaffolds with or without functionalisation with 20 ng/mL FGF2. The percentage of cells that express CD73, CD105, CD90 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, HLA-DR at d) day 9, and e) day 14 post-seeding are shown. f) Representative surface marker expression profiles of MSC populations grown on various substrates for 14 days. Cell samples are shown in blue, while isotype controls are in red. Gatings used to quantify marker expression are also indicated.

Article Snippet: Surface-bound FGF2 was detected with 1 μg/mL mouse monoclonal anti-FGF2 antibody (Abcam) for 1 h at RT and washed three times with PBS.

Techniques: Modification, Staining, Marker, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 1. High FGF19 expression is correlated with liver metastasis in colorectal cancer. A) Flowchart of CRLM-related gene screening using the bioinfor- matics method. Left panel: Venn diagram showing the 33 overlapping DEGs (log2 |fold change|≥2 and p-value < 0.05) in three CRC- and CRLM-related datasets from the Human Genome Array. Right panel: The prognostic value of the 33 overlapping genes was further validated in TCGA CRC cohort. Ten candidates were screened out. B) Among the 10 candidates, FGF19 was confirmed to have the strongest prognostic value. C) Kaplan‒Meier survival curves of progression-free survival for CRC patients with low (n = 247) and high (n = 161) FGF19 expression. Patients were stratified into FGF19-high or FGF19-low groups according to the cutoffvalue. D) Representative IHC staining of FGF19 expression in normal colon epithelium, CRC, and paired primary CRC and CRLM tissues. Scale bar: 50 μm. E) FGF19 expression was higher in CRC tissues (n = 122) than that in normal colon epithelium tissues (n = 122), and higher in LM(+) CRC tissues (n = 12) than that in LM(−) CRC tissues (n = 110). F) FGF19 expression was higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). G) Kaplan‒Meier survival curves of overall and liver metastasis-free survival of CRC patients with low (n = 59) and high (n = 63) FGF19 expression. H) The serological level of FGF19 was higher in patients with LM(+) CRC (n = 12) than that in those with LM(−) CRC (n = 110). I) Kaplan‒Meier survival curves of overall and liver metastasis-free survival of CRC patients with low (n = 71) and high (n = 51) serological levels of FGF19. *p < 0.05, **p < 0.01, ***p < 0.001. In (E) and (H), data were subjected to Mann–Whitney test (data not normally distributed). In (F), data were subjected to paired Student’s t-test. In (B), (C), (G), and (I), data were subjected to Cox proportional hazards regression. See also related Figure S1, Supporting Information.

Article Snippet: Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the concentrations of human FGF19 (DF1900, R&D Systems), IL-1α (DLA50, R&D Systems), IL-1β (DLB50, R&D Systems) and complement C5a (ab193695, Abcam) in culture supernatants or serum as described.

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 2. FGF19 promoted CRLM in a mouse model. A–C) Splenic injection of HCT-15 cells with FGF19 overexpression for liver metastasis experiments (n = 6 mice). A) IVIS images and quantification, B) liver weight and the proportion of tumor metastases in livers, and C) animal overall survival are shown. Scale bar: 1 mm. D–F) Splenic injection of KM12SM cells with FGF19 knockdown for liver metastasis experiments (n = 6 mice). D) IVIS images and quantification, E) liver weight and the proportion of tumor metastases in livers, and F) animal overall survival are shown. Scale bar: 1 mm. G) Mice were administrated intravenously with AAV-shCtrl or AAV-Tbg-shFgf15 (1.0 × 1012 genome copies per mouse) for 4 weeks and then injected FGF19- overexpressing HCT-15 cells for liver metastasis experiments (n = 5 mice). The proportion of tumor metastases in livers is shown. H) Experimental design of rhFGF19 (50 μg kg−1) treatment and sequential splenic injection of HCT-15 cells in a liver metastasis mouse model. Liver specimens (n = 5 mice) were harvested 14 days after splenic injection of HCT-15 cells. Scale bar: 1 mm. I,J) Liver metastasis of mice with splenic injection of FGF19- overexpressing HCT-15 cells treated with or without FGF19 neutralizing antibody (25 μg per mouse) (n = 6 mice). I) Liver weight and the proportion of tumor metastases in livers and J) animal overall survival are shown. *p < 0.05, **p < 0.01, ***p < 0.001; and n.s., nonsignificant. In (A), (B), (D), (E), (G), and (I), data were subjected to Student’s t-test (data normally distributed). In (C), (F), and (J), data were subjected to log-rank test. See also related Figure S2, Supporting Information.

Article Snippet: Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the concentrations of human FGF19 (DF1900, R&D Systems), IL-1α (DLA50, R&D Systems), IL-1β (DLB50, R&D Systems) and complement C5a (ab193695, Abcam) in culture supernatants or serum as described.

Techniques: Injection, Over Expression, Knockdown

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 3. FGF19 mediated the formation of inflammatory CAFs in liver metastatic niches. A,B) mRNA sequencing of LX-2 cells stimulated with 0, 50, or 100 ng mL−1 rhFGF19 for 24 h. A) Bubble plot showing the GO signatures enriched in rhFGF19-stimulated LX-2 cells. B) Heatmap showing the mRNA expression of iCAF and myCAF markers in rhFGF19-stimulated LX-2 cells. C) mRNA expression of iCAF markers in LX-2 cells treated with FGF19- containing CM in the presence or absence of FGF19 neutralizing antibody (10 μg mL−1) for 24 h (n = 6). D) mRNA expression of iCAF markers in LX-2 cells treated with rhFGF19 (50 ng mL−1) for 24 h (n = 6). E) IF staining of iCAFs (PDPN+ and IL-6+ cells) formed by LX-2 cells treated with FGF19- containing CM or rhFGF19 for 48 h. Scale bar: 20 μm. F) IF staining of iCAFs (PDPN+ and IL-6+ cells) in primary CRC and CRLM tissues. Scale bar: 50 μm. G) Correlation between FGF19 expression and the frequency of iCAFs in human CRLM tissues (n = 30). H) Phosphorylation of FGFR4, JAK2, and STAT3 in LX-2 cells treated with rhFGF19 or FGF19-containing CM in the presence or absence of FGF19 neutralizing antibody for 24 h. I) Phosphorylation of FGFR4, JAK2, and STAT3 in LX-2 cells treated with FGF19-containing CM in the presence or absence of fisogatinib (100 nм) or BLU9931 (10 μм). J) Extracellular expression of IL-1𝛼in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM in the presence or absence of fisogatinib or BLU9931 (n = 4). K) mRNA expression of iCAF markers in LX-2 cells treated with FGF19-containing CM in the presence or absence of anakinra (20 mg mL−1) for 24 h (n = 6). L) Phosphorylation of JAK2 and STAT3 in LX-2 cells treated with CM obtained from LX-2 cells pretreated with FGF19-containing tumor cell CM, in the presence or absence of anakinra. M) mRNA expression of iCAF markers in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected

Article Snippet: Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the concentrations of human FGF19 (DF1900, R&D Systems), IL-1α (DLA50, R&D Systems), IL-1β (DLB50, R&D Systems) and complement C5a (ab193695, Abcam) in culture supernatants or serum as described.

Techniques: Sequencing, Expressing, Staining, Phospho-proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 4. FGF19 regulated NET formation in liver metastatic niches to promote the liver metastasis of colorectal cancer. A) IF staining of NETs (H3Cit+ and MPO+ structures) formed by neutrophils treated with CM derived from FGF19-overexpressing HCT-15 and FGF19-knockdown KM12SM cells for 12 h (n = 18 RMFs from 6 experimental replicates per group). Scale bar: 50 μm. B) IF staining and quantification of NETs in livers of mice injected with FGF19-overexpressing HCT-15 and FGF19-knockdown KM12SM cells (n = 18 RMFs from 6 mice per group). Scale bar: 50 μm. C) IF staining of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with CM derived from FGF19-overexpressing HCT-15 cells in the presence or absence of anakinra for 12 h (n = 18 RMFs from 6 experimental replicates per group). Scale bar: 50 μm. D) IF staining and quantification of NETs in the livers of mice injected with FGF19-overexpressing HCT-15 cells with or without anakinra treatment (n = 18 RMFs from 6 mice per group). Scale bar: 50 μm. E–G) Liver metastasis of mice with splenic injection of FGF19-overexpressing HCT-15 cells, with or without DNAse I treatment (5 mg kg−1) (n = 6 mice). IF staining and quantification of NETs in the livers of mice (n = 18 RMFs from 6 mice per group), F) liver weight and the proportion of tumor metastases in livers, and G) animal overall survival are shown. Scale bar: 50 μm. H–J) Liver and serum specimens obtained on the indicated days after

Article Snippet: Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the concentrations of human FGF19 (DF1900, R&D Systems), IL-1α (DLA50, R&D Systems), IL-1β (DLB50, R&D Systems) and complement C5a (ab193695, Abcam) in culture supernatants or serum as described.

Techniques: Staining, Derivative Assay, Knockdown, Injection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 5. FGF19 induced NET formation by facilitating complement C5a and IL-1𝛽production in iCAFs. A) Cytokine array of the media of LX-2 cells pretreated with rhFGF19 (50 ng mL−1) for 24 h. Seven factors were upregulated in the supernatant of LX-2 cells stimulated with rhFGF19. B) Quantification of NETs formed by neutrophils stimulated with complement C5a, CXCL11, IL-1𝛼, IL-1𝛽, IL-18, MIF, or PAI-1 (n = 18 RMFs from 6 experimental replicates per group). C) Intracellular and D) extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with rhFGF19 or FGF19-containing CM (n = 3 for WB, or 6 for ELISA). E) Quantification of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with FGF19-containing CM in the presence of C5a neutralizing antibody (50 ng mL−1) or IL-1𝛽neutralizing antibody (1 μg mL−1) (n = 18 RMFs from 6 experimental replicates per group). F) Representative IHC staining of C5a or IL-1𝛽expression in paired primary CRC and CRLM tissues. Scale bar: 50 μm. G) Expression of complement C5a and IL-1𝛽were higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). H) Correlation between IHC scores of FGF19 and C5a or IL-1𝛽in human CRLM tissues (n = 30). I) ChIP‒qPCR assays showed the recruitment of STAT3 to the promoter regions of C5 and IL1B (n = 3). J,K) Intracellular and L) extracellular expression of C5a and IL-1𝛽in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM and treated with or without fisogatinib (100 nм), fedratinib (10 μм), C188-9 (5 μg mL−1), and anakinra (20 mg mL−1) (n = 3 for WB, or 6 for ELISA). M) Extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected to regular medium, regular medium with rhIL-1𝛼(1 ng mL−1), FGF19-free CM, fresh FGF19-containing CM, or anakinra for 24 h (n = 6). ** or ##p < 0.01; *** or ###p < 0.001; n.s., nonsignificant. In (B), (D), (I), (L), and (M), data were subjected to Student’s t-test. In (E), data were subjected to Mann–Whitney test. In (G), data were subjected to paired Student’s t-test. See also related Figure S6, Supporting Information.

Article Snippet: Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the concentrations of human FGF19 (DF1900, R&D Systems), IL-1α (DLA50, R&D Systems), IL-1β (DLB50, R&D Systems) and complement C5a (ab193695, Abcam) in culture supernatants or serum as described.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 6. NET formation was correlated with FGF19 in liver metastases, and targeting FGF19 signaling with fisogatinib prevented CRLM. A) IF staining and quantification of NETs in primary CRC and CRLM tissues (n = 30). Scale bar: 50 μm. B) Correlation between serological FGF19 and MPO-DNA (n = 122). C) Kaplan‒Meier survival curves of liver metastasis-free survival of patients with CRC with low (n = 72) and high (n = 50) serological levels of MPO-DNA. D–I) Splenic injection of KM12SM and FGF19-overexpressing HCT-15 cells for liver metastasis experiments (n = 6 mice). D) IVIS images and quantification, E) ultrasound images, F) liver weight and the proportion of tumor metastases in livers, G,H) IF staining and quantification of NETs in livers, and I) animal overall survival are shown. Scale bar for H&E: 1 mm. Scale bar for IF: 50 μm. **p < 0.01; ***p < 0.001. In (D), (F), and (H), data were subjected to Student’s t-test. In (A), data were subjected to paired Student’s t-test. In (C), data were subjected to Cox proportional hazards regression. In (I), data were subjected to log-rank test. See also related Figure S7, Supporting Information.

Article Snippet: Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the concentrations of human FGF19 (DF1900, R&D Systems), IL-1α (DLA50, R&D Systems), IL-1β (DLB50, R&D Systems) and complement C5a (ab193695, Abcam) in culture supernatants or serum as described.

Techniques: Staining, Injection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 7. Schematic depiction of the mechanism of FGF19-mediated colorectal cancer liver metastasis via induction of iCAF and NET formation. Created with BioRender.com.

Article Snippet: Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the concentrations of human FGF19 (DF1900, R&D Systems), IL-1α (DLA50, R&D Systems), IL-1β (DLB50, R&D Systems) and complement C5a (ab193695, Abcam) in culture supernatants or serum as described.

Techniques:

Journal: Developmental biology

Article Title: Loss of Fgfr2 leads to partial XY sex reversal.

doi: 10.1016/j.ydbio.2007.11.010

Figure Lengend Snippet: Fig. 7. FGF9 does not bind to the coelomic epithelium of Fgfr2 mutant XY gonads. E11.5 gonad sections were incubated with human FGF9, which was detected with an antibody specific to the human FGF9 protein (green). Human FGF9 bound to the coelomic epithelium in E11.5 XY gonad sections, but not in E11.5 XX and XY Fgfr2 mutant gonad sections. Arrowheads denote the surface of the coelomic epithelium.

Article Snippet: E11.5 frozen sections (12 μm) of XY control, XX control and XY Fgfr2 mutant gonads were treated with human FGF9 protein (0.7 μg/ ml) (R&D systems; AF-273-NA), which was detected by an antibody specific to human FGF9 (R&D systems; 273-F9).

Techniques: Mutagenesis, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Mice

doi: 10.3390/ijms25179308

Figure Lengend Snippet: Mice receiving an adenine-rich diet develop skeletal muscle atrophy. We analyzed C57BL/6J mice that received an adenine-rich or control (CTRL) diet for 14 weeks. ( A ) Blood urea nitrogen (BUN) levels ( n = 5–6; **** p < 0.0001), ( B ) serum phosphate levels ( n = 5–6; ** p < 0.01), and ( C ) serum levels of FGF23 determined by ELISA ( n = 5–6; ** p < 0.01). ( D ) Representative cross-sectional magnetic resonance images (MRI) from the hindlimb (scale bar = 2 mm). ( E ) Quantification of the hindlimb area in mm 2 using MRI images ( n = 1–2). ( F ) Grip strength in newtons (N) normalized to tibia length (TL) in mm ( n = 11–12; ** p < 0.01). ( G ) Gastrocnemius weight (GW) in mg normalized to TL in mm ( n = 11–12; ** p < 0.01). ( H ) Representative immunofluorescence images of gastrocnemius sections stained with anti-laminin to visualize cell borders and with DAPI to visualize cell nuclei (scale bar = 100 μm). ( I ) Quantification of the cross-sectional area of individual myofibers in μm 2 based on anti-laminin immunostainings of gastrocnemius muscle sections ( n = 5; * p < 0.05). ( J – M ) qRT-PCR expression analysis of gastrocnemius (gastro), quadricep (quad), soleus, and tibialis anterior (TA) muscles for Tripartite motif containing 63 ( Trim63 ), F-box protein 32 ( Fbxo32 ), Myostatin ( MSTN ), and Metallothionein 1 ( MT1 ) ( n = 4–5; * p < 0.05, ** p < 0.01). Comparison between CTRL versus adenine mice was performed in an unpaired two-tailed t -test or a one-way ANOVA followed by a post-hoc Tukey’s test. All values are mean ± standard error of the mean (SEM).

Article Snippet: Serum phosphate and BUN biochemistries were analyzed by IDEXX Bioanalytic Laboratories (Columbia, MO, USA), and serum FGF23 was quantified using an ELISA detecting total mouse FGF23 (60-6300, Quidel, San Diego, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Muscles, Comparison, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Mice

doi: 10.3390/ijms25179308

Figure Lengend Snippet: Col4a3 −/− mice develop skeletal muscle atrophy. We analyzed mice with global deletion of Collagen type IV alpha 3 chain (−/−) and wildtype littermates (+/+) at 10 weeks ( A – P ) and 5 weeks ( Q – V ) of age. ( A ) Blood urea nitrogen (BUN) levels ( n = 5–6; *** p < 0.001), ( B ) serum phosphate levels ( n = 5–6; *** p < 0.001), and ( C ) serum levels of FGF23 determined by ELISA ( n = 3–4; * p < 0.05). ( D ) Representative cross-sectional magnetic resonance images (MRI) from the hindlimb (scale bar = 2 mm). ( E ) Quantification of the hindlimb area in mm 2 using MRI images ( n = 1). ( F ) Grip strength in newtons (N) normalized to tibia length (TL) in mm ( n = 7–10; * p < 0.05). ( G ) Gastrocnemius weight (GW) in mg normalized to TL in mm ( n = 9–12; ** p < 0.01). ( H ) Representative immunofluorescence images of gastrocnemius muscle sections stained with anti-laminin to visualize cell borders and with DAPI to visualize cell nuclei (scale bar = 100 μm). ( I ) Quantification of the cross-sectional area of individual myofibers in μm 2 based on anti-laminin immunostainings of gastrocnemius sections ( n = 5; * p < 0.05). ( J – M ) qRT-PCR expression analysis of the gastrocnemius for Tripartite motif containing 63 ( Trim63 ), F-box protein 32 ( Fbxo32 ), Myostatin ( MSTN ), and Metallothionein 1 ( MT1 ) ( n = 6–10; * p < 0.05). ( N ) Representative images of Western blots to analyze the protein expression of TRIM63 and FBXO32 in isolated gastrocnemius tissue. GAPDH serves as a loading control. ( O , P ) Quantification of Western blot signals of TRIM63 and FBXO32 by densitometry ( n = 3–4; ** p < 0.01, **** p < 0.0001). ( Q ) Grip strength of 5-week-old mice in N normalized to TL in mm ( n = 2). ( R ) GW in mg normalized to TL in mm ( n = 8–9). ( S – V ) qRT-PCR expression analysis of the gastrocnemius for Trim63 , Fbxo32 , MSTN , and MT1 ( n = 5–6). Comparison between Col4a3 + / + versus Col4a3 − / − mice was performed in an unpaired two-tailed t -test. All values are mean ± standard error of the mean (SEM).

Article Snippet: Serum phosphate and BUN biochemistries were analyzed by IDEXX Bioanalytic Laboratories (Columbia, MO, USA), and serum FGF23 was quantified using an ELISA detecting total mouse FGF23 (60-6300, Quidel, San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Western Blot, Isolation, Control, Comparison, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Mice

doi: 10.3390/ijms25179308

Figure Lengend Snippet: Mice receiving a high-phosphate diet develop reduced skeletal muscle mass and function but not atrophy. We analyzed C57BL/6J mice that received a 3% phosphate (Pi) or control (CTRL) diet for 3 or 6 months. ( A ) Blood urea nitrogen (BUN) levels ( n = 4–10), ( B ) serum phosphate levels ( n = 5–10; **** p < 0.0001), and ( C ) serum levels of FGF23 determined by ELISA ( n = 5–6; *** p < 0.001). ( D ) Representative cross-sectional magnetic resonance images (MRI) from the hindlimb (scale bar = 2 mm). ( E ) Quantification of the hindlimb area in mm 2 using MRI images ( n = 1–2). ( F ) Grip strength in newtons (N) normalized to tibia length (TL) in mm ( n = 5–10; ** p < 0.01). ( G ) Gastrocnemius weight (GW) in mg normalized to TL in mm ( n = 5–10; * p < 0.05). ( H ) Representative immunofluorescence images of gastrocnemius muscle sections stained with anti-laminin to visualize cell borders and with DAPI to visualize cell nuclei (scale bar = 100 μm). ( I ) Quantification of the cross-sectional area of individual myofibers in μm 2 based on anti-laminin immunostainings of gastrocnemius sections ( n = 4–5). ( J – M ) qRT-PCR expression analysis of the gastrocnemius for Tripartite motif containing 63 ( Trim63 ), F-box protein 32 ( Fbxo32 ), Myostatin ( MSTN ), and Metallothionein 1 ( MT1 ) ( n = 3–8; ** p < 0.01). Comparison between 3-month CTRL and Pi or between 6-month CTRL and Pi was performed using an unpaired two-tailed t -test or a one-way ANOVA followed by a post-hoc Tukey’s test. All values are mean ± standard error of the mean (SEM).

Article Snippet: Serum phosphate and BUN biochemistries were analyzed by IDEXX Bioanalytic Laboratories (Columbia, MO, USA), and serum FGF23 was quantified using an ELISA detecting total mouse FGF23 (60-6300, Quidel, San Diego, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Comparison, Two Tailed Test