Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 6 An increase in levels of acetyl-CoA and H3K27ac promotes CXCL5 secretion in CAFs. The effects of acetate treatment on acetyl-CoA levels in LX-2 cells incubated with ExoHCT116 (A) and ExoSW480 (B). Western blot showed the effects of acetate treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 (C) and ExoSW480 (D). E The effects of Trichostatin A (TSA) treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 KD and their relative control. F The effects of C646 treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoSW480 OE and their relative control. G, H Chromatin immunoprecipitation (ChIP) assays using IgG as a control were performed with antibody against H3K23ac. Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl or in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl and treated with TSA (G). Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl or in LX- 2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl and treated with C646 (H). Each experiment was performed in triplicate. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Incubation, Western Blot, Control, Chromatin Immunoprecipitation

Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 8 A schematic diagram of mechanism of exosomal HSPC111 facilitates CRLM via reprogramming lipid metabolism in CAFs. Exosomal HSPC111 derived from CRC cells phosphorylates ACLY in CAFs, leading to the increased levels of acetyl-CoA and histone acetylation to secrete CXCL5, and resulting in CRC cells colonized in liver via the CXCL5-CXCR2 axis.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Oligonucleotide primer sets for real-time PCR.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Expressions of CXCL6, CXCR1, and CXCR2 in osteosarcoma (OS) cells. The mRNA expressions of CXCL6 (A) , CXCR1 (B) , and CXCR2 (C) in multiple OS cell lines were evaluated by real-time PCR. The protein levels of CXCL6 (D) , CXCR1 (E) , and CXCR2 (F) in multiple OS cell lines were detected by western blot assay. (G–I) The protein quantification histograms were shown.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Anti-CXCL6 antibody inhibited the migration, invasion and EMT of OS cells. OS cells were cultured for 72 h, then incubated with anti-CXCL6 antibody (10 μg/mL) or a control antibody (IgG, 10 μg/mL) for another 24 h. (A) The level of CXCL6 in the supernatant fluid of cultured MG63 and 143B cells was detected by ELISA. (B) The migration of MG63 and 143B cells was evaluated by Transwell assay (no matrigel). Scal bar = 100 μm. (C,D) The number of migrated cells was shown. (E) The invasion of MG63 and 143B cells was assessed by Transwell assay (matrigel). Scal bar = 100 μm. (F,G) The number of invasive cells was shown. The expressions of E-cadherin (H) and N-cadherin (I) in MG63 and 143B cells were determined by immunofluorescence assay. Scal bar = 50 μm. (J) The protein levels of E-cadherin, N-cadherin, and Snail were evaluated by western blot assay. (K–P) The protein quantification histograms were shown. (Q–S) The mRNA expression of E-cadherin, N-cadherin, and Snail was detected by real-time PCR. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, versus the OS cell group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Migration, Cell Culture, Incubation, Control, Enzyme-linked Immunosorbent Assay, Transwell Assay, Immunofluorescence, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Recombinant human (rh) CXCL6 facilitated the migration and invasion of OS cells. OS cells were treated with 100 ng/ml rhCXCL6 for 24 h. (A) The migration of SaOS-2 and U2OS cells was detected by Transwell assay (no matrigel). Scal bar = 100 μm. (B,C) The number of migrated cells was shown. (D) The invasion of SaOS-2 and U2OS cells was determined by Transwell assay (matrigel). Scal bar = 100 μm. (E,F) The number of invasive cells was shown. ∗∗ P < 0.01, ∗∗∗ P < 0.001, versus the OS cell group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Recombinant, Migration, Transwell Assay

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: CXCL6/CXCR2 axis contributed to migration and invasion of OS cells. After transfection with siRNAs for 24 h, OS cells were treated with 100 ng/ml rhCXCL6 for 24 h. The mRNA expression of CXCR2 in SaOS-2 (A) and U2OS (B) cells was detected by real-time PCR. The protein expression of CXCR2 in SaOS-2 (C) and U2OS (D) cells was assessed by western blot assay. The protein quantification histograms were shown. (E) The migration of SaOS-2 and U2OS cells was detected by Transwell assay (no matrigel). Scal bar = 100 μm. (F,G) The number of migrated cells was shown. (H) The invasion of SaOS-2 and U2OS cells was determined by Transwell assay (matrigel). Scal bar = 100 μm. (I,J) The number of invasive cells was shown. (K) The protein levels of MMP9 and Snail in SaOS-2 and U2OS cells were detected by western blot assay. (L–O) The protein quantification histograms were shown. (P) MMP-9 activity in the supernatant fluid of cultured SaOS-2 and U2OS cells was determined by gelatin zymography method. (Q,R) The quantification histograms were shown. ∗∗ P < 0.01, ∗∗∗ P < 0.001, versus the SaOS-2+NC or U2OS+NC group. # P < 0.05, ## P < 0.01, ### P < 0.001, versus the rhCXCL6+NC group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Migration, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transwell Assay, Activity Assay, Cell Culture, Zymography

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Effect of CXCL6/CXCR2 axis on E-cadherin and N-cadherin expressions. The expressions of E-cadherin (A) and N-cadherin (B) in SaOS-2 and U2OS cells with different treatments were determined by immunofluorescence assay. Scal bar = 50 μm.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: PI3K/AKT and β-catenin signaling pathways participated in the regulation of migration, invasion and EMT by CXCL6/CXCR2 axis in OS cells. After transfection with siRNAs for 24 h, OS cells were treated with 100 ng/ml rhCXCL6 for 24 h. (A) The protein levels of p-AKT, AKT, and nuclear β-catenin in SaOS-2 and U2OS cells were detected by western blot assay. β-actin and Histone H3 were used as loading controls. (B–E) The protein quantification histograms were shown. OS cells were pre-treated with 50 μM LY294002 or 10 μM XAV939 for 1 h, then treated with 100 ng/ml rhCXCL6 for 24 h. (F) The migration of SaOS-2 and U2OS cells was detected by Transwell assay (no matrigel). Scal bar = 100 μm. (G,H) The number of migrated cells was shown. (I) The invasion of SaOS-2 and U2OS cells was determined by Transwell assay (matrigel). Scal bar = 100 μm. (J,K) The number of invasive cells was shown. (L) The protein levels of E-cadherin, N-cadherin, Snail, and MMP9 in SaOS-2 and U2OS cells were detected by western blot assay. (M–T) The protein quantification histograms were shown. (U) MMP-9 activity in the supernatant fluid of cultured SaOS-2 and U2OS cells was assessed by gelatin zymography assay. (V,W) The quantification histograms were shown. ∗∗∗ P < 0.001, versus the OS cell or OS cell +NC group. # P < 0.05, ## P < 0.01, ### P < 0.001, versus the rhCXCL6+NC or rhCXCL6 group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: Protein-Protein interactions, Migration, Transfection, Western Blot, Transwell Assay, Activity Assay, Cell Culture, Zymography Assay

Journal: Frontiers in Pharmacology

Article Title: Activation of CXCL6/CXCR1/2 Axis Promotes the Growth and Metastasis of Osteosarcoma Cells in vitro and in vivo

doi: 10.3389/fphar.2019.00307

Figure Lengend Snippet: Effect of CXCL6/CXCR2 axis on OS tumor growth and pulmonary metastasis in vivo . U2OS cells were infected with lentivirus expressing CXCL6 or NC. The mRNA (A) and protein level (B) of CXCL6 in U2OS cells were determined by real-time PCR and western blot assay. (C) After the injection of LV-CXCL6 or LV-NC U2OS cells for 21 days, the representative images of nude mice and their removed xenograft tumors were shown. (D) The tumor volume was calculated and the tumor growth-curve was shown. (E) The weight of xenograft tumors was shown. (F) The expression of PCNA in tumor tissues was detected by immunohistochemical staining. Scal bar = 50 μm. (G) The percentage of PCNA positive cells were calculated and shown. (H) The protein levels of p-AKT, AKT, and nuclear β-catenin in tumor tissues were detected by western blot assay. β-actin and Histone H3 were used as loading controls. (I,J) The protein quantification histograms were shown. (K) Representative images of the lung of nude mice. (L) The number of lung metastatic nodes was quantified. ∗∗∗ P < 0.001, versus the LV-NC group. ## P < 0.01, ### P < 0.001, versus the LV-CXCL6 group.

Article Snippet: The CXCL6 level in the supernatant fluid of cultured OS cells was determined by a CXCL6 ELISA kit (BOSTER, Wuhan, China) according to the manufacturer’s protocol.

Techniques: In Vivo, Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Immunohistochemical staining, Staining

Journal: The FASEB Journal

Article Title: Genome-wide effect of pulmonary airway epithelial cell–specific Bmal1 deletion

doi: 10.1096/fj.201801682R

Figure Lengend Snippet: Cell-autonomous Bmal1 function in ALI culture of primary mouse tracheal cells. ALI cultures were established using primary tracheal epithelial cells from WT and global Bmal1−/− mice, with 7 d under submerged condition and another 10 d exposed to air in the apical side of cells. A, B) Electron microscopy images for cilia in ALI primary tracheal cells from WT and global Bmal1−/− mice, respectively. C, D) Immunofluorescence staining of cilia markers (acetylated tubulin) and epithelial cell markers (E-cadherin) in ALI primary tracheal cells from WT and global Bmal1−/− mice, respectively. E, F) Cxcl5 and Cxcl15 gene expression measurement by qPCR in ALI primary tracheal cells from WT and global Bmal1−/− mice. Data shown are means ± sd; Student’s t test (n = 3/genotype). *P < 0.05, **P < 0.01. G) Expression of Bmal1 exon 8 in primary tracheal cells 2 d after infection. Data shown are means ± sd; Student’s t test (n = 3/genotype). ***P < 0.001. H) Bioluminescence traces of Per2-luc in control and Adv-Cre–transfected ALI primary tracheal cells 21 d in culture. I) Schematic description of viral Cre infection in primary tracheal cells from Bmal1flox/flox mice. J) Overlap of expression of rhythmic genes during ALI culture and LCM studies. K) KEGG pathway enrichment of DE genes in cell culture study. Pathways are ranked by significance value.

Article Snippet: CXCL5 levels were measured with a CXCL5 ELISA Kit (DY254; Bio-Techne, Minneapolis, MN, USA), according to product protocol.

Techniques: Electron Microscopy, Immunofluorescence, Staining, Expressing, Infection, Transfection, Cell Culture

Journal: The FASEB Journal

Article Title: Genome-wide effect of pulmonary airway epithelial cell–specific Bmal1 deletion

doi: 10.1096/fj.201801682R

Figure Lengend Snippet: Reverse feeding resets time of day variation in pulmonary LPS response. A) Schematic description of experiment design. The food-reversal experiment was undertaken using 2 separate cohorts. B–D) Food reversal without LPS treatment was performed in cohort 1. E–G) Aerosolized LPS exposure experiment at ZT0 vs. ZT12 was performed in cohort 2 and total BAL cells, neutrophils, and CXCL5 concentrations measured. Gene expression was determined in cohort 2. Data analyzed by 2-way ANOVA with post hoc test to examine time of day difference within genotypes (n = 6–8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (significant time of day difference within genotypes).

Article Snippet: CXCL5 levels were measured with a CXCL5 ELISA Kit (DY254; Bio-Techne, Minneapolis, MN, USA), according to product protocol.

Techniques: Expressing