Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: MCPyV truncated LT induces paranuclear retention and stabilization of KIT. (A) Immunofluorescence detection of KIT (green) and MCPyV LT (CM2B4, red) or sT (CM8E6, red) in KIT-HEK293 cells transfected with MCPyV T-antigens or vector control ( n = 6). (B) Top : illustration of the expression constructs of LT339 and the VPS39-interaction defective mutant LT339 W209A . Bottom : Representative images showing the effect of LT339 and LT339 W209A mutant on localization of KIT (green). LT was detected by CM2B4 (red). ( n = 3) (C) Immunoblots showing the effect of the LT339 and LT339 W209A on KIT expression. The quantification of KIT level is shown below the immunoblots ( n = 9). (D) Top : immunoblot analysis of the effect LT339 and LT339 W209A on KIT protein stability in the presence of cycloheximide (CHX) up to 4 h. Bottom : quantification of KIT protein stability after normalization to 0 h time point ( n = 4). (E) Immunoblots showing KIT protein stability in KIT-HEK293 cells transfected with LT339, LT339 W209A or plasmid control (CTR) treated with KIT ligand (KITLG, 100 ng/mL) or solvent control (PBS) in the presence of CHX (100 μg/mL). (F) Quantification of KIT protein stability after normalization to 0 h time point ( n = 5). Solid lines represent PBS control (KITLG − ) and dotted lines represent KITLG treatment (KITLG + ). (A and B) Nuclei were stained by DAPI (blue). Scale bar: 10 μm. Numbers below the images refer to the proportion of cells with KIT paranuclear dot-like staining to the total number of cells analyzed. (C, D and F) Error bars represent mean ± SEM. * p < 0.05, *** p < 0.001, ns = not significant were calculated by one-way ANOVA with post-hoc Tukey’s test (C), two-way ANOVA (D) or two-way ANOVA with post-hoc Bonferroni’s test (F).

Article Snippet: For KITLG induced KIT degradation experiments, KIT-293 cells were transfected with LT339, LT339 W209A or CTR for 48 h, followed by addition of CHX (100 μg/mL) and KITLG (100 ng/mL; Proteintech Group, HZ-1024) or PBS.

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Control, Expressing, Construct, Mutagenesis, Western Blot, Solvent, Staining

Journal: Scientific Reports

Article Title: Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level

doi: 10.1038/s41598-017-01619-1

Figure Lengend Snippet: Caveolin-1 as a Potential Mediator of FlgE Stimulation via the MEK-Erk Pathway. ( A ) Confirmation of CAV1 presence among HCEC membrane proteins and among proteins pulled down by the FlgE or FlgEM-conjugated resin. The pull-down procedure was also performed with a “resin” control that was not conjugated with FlgE. ( B ) Impairment of IL1β and Cxcl1 expression induced by FlgE (5 μg/mL), the flagellar hook (5 μg/mL) or LPS (5 μg/mL) in organoid cultures of CAV1 −/− mice lungs compared with that in WT mouse lungs. Please note that IL6 expression induced by these agonists was not hindered by CAV1 deficiency. RT-qPCR was performed using TaqMan probes, and the mRNA expression level of each gene is relative to the expression level of that gene in WT mice. Data are representative of two independent experiments. Three mice were used for making lung slices that were evenly distributed into each treatment wells, and two duplicate wells were set for each treatment. *P < 0.05, **P < 0.01, ***P < 0.001 between WT and CAV1 −/− . ( C ) Involvement of Erk and Stat3 pathways in FlgE stimulation in organoids that were blocked by CAV1 deficiency. For densitometry, the intensity of each gene at each time point was normalized against sample at 0 minute. Data are presented as the means ± SEM, n = 2. ( D ) A unique MEK inhibitor, U0126 (10 µM), was added to WT organoids 30 min before the addition of FlgE to further confirm the involvement of the MEK-Erk pathway in FlgE stimulation. Data are representative of three independent experiments that yielded similar results. N = 3 mice for each group of lung organoid cultures. Two samples were analyzed in each group. *P < 0.05, **P < 0.01, ***P < 0.001, between WT and CAV1 −/− .

Article Snippet: Cytokines in the lavage were measured using ELISA kits for murine IL1β, IL6 and CXCL1 (Cusabio Biotech, Wuhan, China), and serum MPO activity was measured using an MPO determination kit (Nanjing Jiancheng Corp., Nanjing, China).

Techniques: Membrane, Control, Expressing, Quantitative RT-PCR

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 1 CXCL17 is upregulated in lung metastatic breast cancer cells. a Scheme of the animal model. 4T1 cells (500,000 cells per fat pad) were implanted into the mammary fat pads and allowed to spontaneously metastasize to the lung for 24 days. Whole primary tumor in mammary or tumor nodules in the lungs (n = 3), livers (n = 4), and intestine (n = 4) and lymph nodes (tumor nodules = 7) were subjected to gene profiling. b Specific gene profile of breast cancer metastasized to lung. c The upregulation of CXCL17 protein in lung metastatic 4T1 cells. 4T1 cells were implanted into mice in an orthotic model. The expression of CXCL17 protein of 4T1 cells isolated from primary sites (mammary) or lung tissue (six pairs of 4T1 cell isolated from in mammary and lung) was assessed by ELISA after 48-h incubation. d In the left panel, the box plot generated from Kao Huang Breast GSE20685 dataset of SurvExpress showed the amount of CXCL17 expression in each group. The right panel showed the Kaplan-Meier time to metastasis curve. e The results were from Van De Vijver Nature 2002 dataset of SurvExpress. The left and right panels showed CXCL17 expression and the Kaplan-Meier time to metastasis curve respectively. The group was divided according to the “Maximize Risk Groups” in SurvExpress website. f Kaplan-Meier distant metastasis-free survival via KM plotter database. High and low CXCL17 expression groups were divided according to “Auto select best cutoff” in the KM plotter website. Each value is the mean ± SD of three determinations; *p < 0.05

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Animal Model, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Incubation, Generated

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 2 CXCL17 increased lung metastasis in vivo. a CXCL17 increased the lung metastasis of 4T1 cells in an orthotropic model. The total number of tumor nodule per whole lung lobes was counted and averaged among the animals of each group. b The H&E staining of tumor sections. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). 4T1 were implanted into the fat pads of mice. Tumor nodules of 4T1 in the primary site and lungs were collected after 24 days of injections. c CXCL17 increased lung metastasis in human breast MDA-MB-231. Nude mice were treated with PBS or recombinant CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). MDA-MB-231 cells were implanted into mice by tail vein injection. The MDA- MB-231 tumor nodules in the lungs of mice were collected after 90 days of injections. d The H&E staining of tumor sections. e Knockdown of CXCL17 decreased the spontaneous metastatic ability of L4T1 cells. f The H&E staining of tumor sections. CXCL17-knockdown-L4T1 were implanted into the fat pads of mice (n = 6 per group). Tumor nodules in the lungs were collected after 24 days of injections. Representative lung tissue sections were stained with H&E and photographed at × 100 magnification. Each value is the mean ± SEM; Mann-Whitney U test was performed, *p < 0.05. T tumor

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: In Vivo, Staining, Recombinant, Injection, Knockdown, MANN-WHITNEY

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 3 CXCL17 increases the recruitment of MDSCs in metastatic lungs of mice. The effect of CXCL17 in the recruitment of CD11b+Gr-1+ MDSCs (a), CD11b+Gr-1−MDSCs (b), and CD11b+F4/80+ macrophages (c) in the lungs of mice. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). Various immune cells were isolated from the lungs of mice by antibody conjugated magnetic beads. Each value is the mean ± SEM; *p < 0.05. CXCL17 increased the migration (d) and transendothelial migration (e) of CD11b+Gr-1+ MDSCs in vitro. GPR35 inhibitor decreased the migration (f) and transendothelial migration (g) of CD11b+Gr-1+ MDSCs induced by CXCL17. CD11b+Gr-1+ MDSCs were isolated from the lungs of normal mice (n = 3). PKH26-labeled CD11b+Gr-1+ MDSCs cells were seeded onto inserts (1 × 105 cells in 3-μm pore insert for migration analysis). For transendothelial migration analysis, C166 cells were seeded in 3-μm pore collagen-coated inserts for confluent monolayer, and PKH26-labeled CD11b+Gr-1+ MDSCs cells (1 × 105/insert) were seeded onto C166 confluent monolayer inserts, and the migration of cancer cells was assessed by fluorescence microscope. CXCL17 (1 ng/ml) were added in bottom well as chemoattractant. For blocking experiment, GPR35 inhibitor (CID2745687, 2 μM) was added in the inserts. Results are representative of at least three independent experiments, and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Recombinant, Isolation, Magnetic Beads, Migration, In Vitro, Labeling, Fluorescence, Microscopy, Blocking Assay

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 4 CXCL17 increases angiogenesis in lung metastatic niche by recruiting CD11b+Gr-1+ MDSCs. a CXCL17 increased CD31+ cells in the lungs of mice. Digital images of tissues were captured and analyzed with ImageJ software to calculate the percentage of positive cells (high positive + positive + low positive cells). b Conditioned medium (CM) (50%) of CD11b+Gr-1+ MDSCs isolated from the lungs of CXCL17-treated mice (n = 5) increased tube formation of C166 cells. The effect of CXCL17 in the PDGF-AA (c), PDGF-BB (d), VEGF-A (e), and EGF basic (f) secretions of CD11b+Gr-1+ MDSCs in vivo. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/ week, n = 6 per group). The lungs of these mice were harvested and constrained by CD31 antibody and H&E. Alternatively, CD11b+Gr-1+ MDSCs were isolated from lungs of PBS or CXCL17 treated mice (n = 6 per group) by antibody conjugated magnetic beads, and CM was collected after culturing for 24 h. The expression of various angiogenic factors was assessed by Luminex Assays. g CXCL17 increased the expression of PDGF-BB in CD11b+Gr- 1+ MDSCs isolated from the lungs of normal mice. CD11b+Gr-1+ MDSCs were isolated from the lungs of normal mice (n = 5) by antibody conjugated magnetic beads and treated with rmCXCL17 (10 ng/ml) for 24 h. The expression of PDGF-BB was assessed by Luminex Assays. h Inhibition of PDGFR-β by specific inhibitor (DMPQ-2HCl, 5 μM) prevents CD11b+Gr-1+ MDSC-mediated C166 tube formation. Results are representative of at least three independent experiments in vitro studies, and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Software, Isolation, In Vivo, Recombinant, Magnetic Beads, Expressing, Luminex, Inhibition, In Vitro

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 5 CD11b+Gr-1+ myeloid cells in lungs metastatic niche promote cancer cell colonization. a CXCL17 increased cancer cell extravasation into the lungs of mice. Athymic nude mice were treated with recombinant CXCL17 protein (1 μg/mouse, 2 times/week, n = 6 per group) for 14 days; MDA-MB-231-RFP-Luc cells were injected into mice by tail vein. The lungs of these mice were harvested after 48-h injection, then examined by a confocal microscope. b Scheme of CD11b+Gr-1+ depletion in the animal model. Depletion of CD11b+Gr-1+ cells decreased CXCL17-mediated cancer extravasation (c) and tumor nodules formation (d) in the lungs of mice. e The H&E staining of tumor sections of lungs. For MDSC depletion studies, mice (n = 6 per group) were treated with isotype or anti-Gr-1 antibodies (Bio X Cell) at 100 μg/mouse intraperitoneal injections every 4th day, and 4T1 cells were injected via tail vein into the mice (n = 6 per group). The control group received intraperitoneal injection of purified rat immunoglobulins. PKH26-labeled 4T1 cells were injected into mice by tail vein for indicated times (24 days for lung metastasis and 48 h for extravasation). The lungs of these mice were harvested and examined by a confocal microscope. Each value is the mean ± SEM; *p < 0.05. White arrows indicate cancer cells

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Recombinant, Injection, Microscopy, Animal Model, Staining, Control, Purification, Labeling

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 6 CD11b+Gr-1+ MDSCs increased breast cancer extravasation and survival via PDGF-BB. CM of CD11b+Gr-1+ MDSC isolated from the lungs of CXCL17-treated or 4T1-bearing mice (n = 5) increased transendothelial migration (a) and colony formation (b) of 4T1 cells. rmPDGF enhanced 4T1 cell transendothelial migration (c) and colony formation (d). Blockade of PDGFR-β prevented transendothelial migration (e) and colony formation (f) of 4T1 cells induced by CM of CD11b+Gr-1+ MDSCs. CD11b+Gr-1+ MDSC isolation and the collection of their CMs have been described in the legend of Fig. 2. C166 cells were seeded 8-μm pore collagen-coated inserts for confluent monolayer, and PKH26-labeled 4T1 cells (1 × 105/insert) were seeded onto C166 confluent monolayer inserts, and the CM of CD11b+Gr-1+ MDSCs (50%) or rmPDGF-BB protein (20 ng/ml) were placed in the bottom well as chemoattractant. The migration of cancer cells was assessed by a fluorescence microscope. For colony formation analysis, 4T1 cells were treated with different CMs (50%) or PDGF-BB (20 ng/ml), and the colonies counted after staining. g PDGFR inhibitor imatinib decreased lung metastasis in mice (n = 6 per group). h H&E staining of lung sections. Representative lung tissue sections were stained with H&E and photographed at × 100 magnification. Results are representative of at least three independent experiments and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Isolation, Migration, Labeling, Fluorescence, Microscopy, Staining

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 7 A novel mechanism underlying the contribution of primary cancer to lung metastatic niche formation in breast cancer. Primary cancer- secreted CXCL17 increases the accumulation of CD11b+Gr-1+ MDSCs in the lungs, which produce PDGF-BB, resulting in enhanced angiogenesis in the lung tissue before cancer cells’ arrival. In addition, CXCL17 also drives CD11b+Gr-1+ MDSCs to exhibit supportive activity for cancer extravasation and survival by PDGF-BB production under cancer cell arrival

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: PKD2/3 enhance MCs migration through upregulation of SCF, CCL5 and CCL11 in prostate cancer cellsmRNA and the protein level of the scf , ccl5 and ccl11 were analyzed by real-time qPCR and ELISA in DU145 ( a ) and PC-3 M ( b ) cells transfected with siRNA of PKD2, PKD3. Data representing the means ± S.D. of three independent experiments was analyzed by one-way ANOVA for significance versus si-CTL. ***p < 0.001, **p < 0.01, *p < 0.05 versus si-CTL. c Knockdown efficiency of PKD2 and PKD3 in prostate cancer cells was verified by Western blotting. d DU145 cells were transfected with siRNA of PKD2, PKD3, the Conditional medium (CM) was collected to measure migration of P815 cells in response to SCF, CCL5, and CCL11 treatment by transwell assay. e Quantification were analyzed from data in D. ***p < 0.001 versus si-CTL by One-ANOVA tests

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown, Western Blot, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: PKD2 and PKD3 promote SCF, CCL5 and CCL11 expression through Erk1/2 signaling pathways. a-b Interaction of PKD2 or PKD3 with Erk1/2 was performed by co-IP assay in PC-3 M or DU145 prostate cancer cells. c-d DU145 cells ( c ) or PC-3 M cells ( d ) were transfected as indicated and treated with 100 nM PMA, phosphorylation and protein expression were detected by western blotting. e Overexpression efficiency of PKD2 and PKD3 in prostate cancer cells was verified by Western blotting. f ELISA were applied to measure SCF in conditional medium from DU145 cell transfected with GFP, GFP-PKD2, and GFP-PKD3 in present with or without Erk inhibitor PD98059(PD) treatment. g Real-time PCR was performed to analyze ccl5 expression in DU145 transfected with GFP, GFP-PKD2 and GFP-PKD3 plasmids followed by treatment with or without Erk inhibitor PD98059

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: Expressing, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Phospho-proteomics, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: PKD2 and PKD3 are required for SCF, CCL5, and CCL11 transcription. a Analysis of the binding site of p65(highlight in red) and AP1(highlight in blue) in the promoters of scf, ccl5 and ccl11 using UCSC software online. b Western blotting was used to ensure the knockdown effect. ChIP analysis of the binding of c-Jun ( c ), c-Fos (d) and NF-κB ( e ) to the scf, ccl5 and ccl11 gene promoter in PC-3 M cells depleted with siRNA of PKD2, PKD3. Student’s t-test , *p < 0.05, **p < 0.01 and ***p < 0.001 ( n = 3). Error bars indicate mean ± S.D.

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: Binding Assay, Software, Western Blot, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: CRT0066101 reduces MCs recruitment and tumor angiogenesis in vivo. a Experimental setting. b C57BL6 mice bearing RM1 tumors were administered a daily vehicle [control group; 5% ( w / v ) dextrose] or CRT0066101 at 20 μM and 40 μM for 2 weeks (4 mice per group), then excised tumor images. c Tumor volume were represented at indicated day. d Immunohistochemistry staining for phospho-PKD, microvessel density (stained with CD31), and mast cells (stained with c-Kit). Representative image were shown in 400X under the microscope (Left panel). Quantification of the indicated parameter was analyzed among groups after treatment with CRT0066101 (Right panel). e Schematic model of the mechanistic role of PKD2 and PKD3 in tumor angiogenesis by regulating SCF-, CCL5-, and CCL11-mediated mast cell recruitment

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: In Vivo, Control, Immunohistochemistry, Staining, Microscopy