Journal: BMC Cancer

Article Title: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

doi: 10.1186/s12885-023-11170-0

Figure Lengend Snippet: RTK/KRAS and VEGF-A/VEGFR-2 inhibition block GA progression in Tcon mice. A Representative H&E photos of Tcon3077 organoids following transduction with VEGF-A shRNA (sh.VEGF-A) and/or KRAS shRNA (sh.KRAS) or control scramble shRNA. Graph showing number of organoids greater than 100m. B Immunofluorescence (IF) photos showing CD44 (green) and cell nuclei (DAPI). Graph showing the percent of DAPI positive cells with expression of the cancer stem cell maker, CD44 in transduced Tcon3077 organoids. C Survival curve of Tcon mice treated with an oral MEK inhibitor, DC101, both MEK inhibitor and DC101, or control ( n = 5–7 per group). D Representative photos of H&E slides for stomach mouse tumors. E – F IHC and graphs for CD44 and CD31 expression in mouse stomach tumors. Bars represent standard deviation. * p < 0.05 compared to control. ** p < 0.05 compared to all other groups

Article Snippet: Immunohistochemistry was performed for patient and mouse tumor samples using the following primary antibodies: CD31 (MA5-13,188; Thermo Fisher Scientific), CD44 (NBP1-31,488; NOVUS Biological USA), p-MEK1/2 (#9154; Cell signaling), YFP (MBS833304; MYBIOSource), p-ERK1/2 (#9101; Cell signaling), VEGF-A (sc-7269; Santa Cruz), and LYVE-1 (AF2089, AF2125; R&D system).

Techniques: Inhibition, Blocking Assay, Transduction, shRNA, Control, Immunofluorescence, Expressing, Standard Deviation

Journal: BMC Cancer

Article Title: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

doi: 10.1186/s12885-023-11170-0

Figure Lengend Snippet: Inhibition of RTK-RAS and VEGF-A/VEGFR2 blocks tumorigenesis in an orthotropic model of GA. A Western blot analysis for KRAS, VEGF-A and β-actin in CD44( +) Tcon3077 cells stably transduced with KRAS shRNA (sh.KRAS), VEGF-A shRNA (sh.VEGF-A) and/or control scramble shRNA (Original version in Suppl. Figs. S B-C). B IVIS images 10 weeks after intra-gastric injection of CD44( +) Tcon3077 cells transduced with sh.KRAS and/or sh.VEGF-A. Graph showing bioluminescence for each group. C Representative gross photos and H&E slides of stomach tumors in mice sacrificed at 12 weeks. D IF images of gastric tumors stained for CD44 (red), CD31 (white), and p-MEK1/2 (green). Graphs showing number of positive cells. E Survival curve of 4 groups of mice. Dashed line represents time of sacrifice (12weeks) of two mice for stomach tissue analysis. Bars represent standard deviation. * p < 0.05 compared to control. ** p < 0.05 compared to all other groups

Article Snippet: Immunohistochemistry was performed for patient and mouse tumor samples using the following primary antibodies: CD31 (MA5-13,188; Thermo Fisher Scientific), CD44 (NBP1-31,488; NOVUS Biological USA), p-MEK1/2 (#9154; Cell signaling), YFP (MBS833304; MYBIOSource), p-ERK1/2 (#9101; Cell signaling), VEGF-A (sc-7269; Santa Cruz), and LYVE-1 (AF2089, AF2125; R&D system).

Techniques: Inhibition, Western Blot, Stable Transfection, Transduction, shRNA, Control, Injection, Staining, Standard Deviation

Journal: BMC Cancer

Article Title: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

doi: 10.1186/s12885-023-11170-0

Figure Lengend Snippet: RTK/KRAS and VEGF-A/VEGFR-2 inhibition CD44( +) GA cells blocks metastasis. A Representative H&E photos of mouse lungs 3.5 weeks after tail vein injection of CD44( +), CD44(-), and unsorted Tcon3077 cells. Graph showing number of metastases > 100m per field for each group. B Graph showing bioluminescence after IVIS imaging of mouse lungs 3.5 weeks after tail vein injection of Tcon3077 cells stably transduced with KRAS shRNA, VEGF-A shRNA, or both. Scramble shRNA was used as a control. C H&E and IF photos (YFP, yellow; DAPI, blue) of mouse lungs. Graphs show number of metastatic foci > 100m per field and percent of YFP-positive cells. D H&E, VEGF-A IHC, and VEGF-A (green)/CD31 (white)/CD44 (red) IF. Graphs showing the percent of CD44, VEGF-A, and CD31-expressing cells. Bars represent standard deviation. * p < 0.05 compared to control. ** p < 0.05 compared to all other groups

Article Snippet: Immunohistochemistry was performed for patient and mouse tumor samples using the following primary antibodies: CD31 (MA5-13,188; Thermo Fisher Scientific), CD44 (NBP1-31,488; NOVUS Biological USA), p-MEK1/2 (#9154; Cell signaling), YFP (MBS833304; MYBIOSource), p-ERK1/2 (#9101; Cell signaling), VEGF-A (sc-7269; Santa Cruz), and LYVE-1 (AF2089, AF2125; R&D system).

Techniques: Inhibition, Injection, Imaging, Stable Transfection, Transduction, shRNA, Control, Expressing, Standard Deviation

Journal: BMC Cancer

Article Title: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

doi: 10.1186/s12885-023-11170-0

Figure Lengend Snippet: Expression of CD44, KRAS, and VEGFA in GA patient tumors. A Gene expression of CD44, KRAS, and VEGF-A in gastric tumors compared to adjacent normal tissues. B Hierarchical clustering of gene expression in 83 patient tumors based on the log 2 fold-change in gene expression in tumor compared to adjacent normal tissue. Clinical information, including tumor location, Lauren classification, TNM stage, and the molecular classifications by Splicing, ACRG, and TCGA methods are indicated for the three clusters defined by CD44 expression (low, intermediate, and high). C Box plots of KRAS and VEGFA expression in each CD44 patient cluster. D Kaplan–Meier overall survival curves stratified by expression of VEGF-A in the FMUUH cohort

Article Snippet: Immunohistochemistry was performed for patient and mouse tumor samples using the following primary antibodies: CD31 (MA5-13,188; Thermo Fisher Scientific), CD44 (NBP1-31,488; NOVUS Biological USA), p-MEK1/2 (#9154; Cell signaling), YFP (MBS833304; MYBIOSource), p-ERK1/2 (#9101; Cell signaling), VEGF-A (sc-7269; Santa Cruz), and LYVE-1 (AF2089, AF2125; R&D system).

Techniques: Expressing, Gene Expression

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: SRGN promotes NSCLC cell migration in a CD44-dependent manner . a-b Wound healing assay was performed by seeding NSCLC-H460 ( a ) and -H1299 ( b ) cells in the Culture-Insert 2 Well μ-Plates. After removing the insert, wound closure was determined at 0, 24, 48, and 72 h. Wound area was assessed by ImageJ and normalized with respect to the area at 0 h. c H460/sh-CTRL and H460/sh-SRGN cells were cultured in serum-free medium for 24 h and subjected to Boyden chamber migration assay. Migrated cells were counted in 3 h. d H1299/Mock and H1299/SRGN cells were sorted based on CD44 expression. The unsorted as well as CD44-negative H1299/Mock and H1299/SRGN cells were cultured in serum-free medium for 24 h, and subjected to Boyden chamber migration assay. The migrated cells were counted in 3 h. e The parental H1299 cells and the Control-KO and CD44-KO cell clones generated using the CRISPR/Cas9 system were subjected to Boyden chamber migration assay as described above. The expression of CD44 was shown by western blot. f CD44 expression in HuTu80/Mock and HuTu80/CD44 cells was assessed by flow cytometry (left panel) and western blot (right panel). g-h HuTu80/Mock and HuTu80/CD44 cells were suspended in conditioned media derived from H1299/Mock and H1299/SRGN cells ( g ) as well as from H460/sh-CTRL and H460/sh-SRGN cells ( h ), and subjected to Boyden chamber migration assay as described above. The Boyden chamber assays were performed by loading the lower chamber with medium containing 5% FBS in ( c ) and ( h ), and 2% FBS in ( d ), ( e ) and ( g ). Data are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Migration, Wound Healing Assay, Cell Culture, Expressing, Clone Assay, Generated, CRISPR, Western Blot, Flow Cytometry, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: CS-GAG modification is critical for SRGN-mediated cell migration. a Western blot analysis of SRGN in the total cell lysate and CM of H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells cultured in serum-free medium for 48 h. b Wound healing assay was performed in H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells. After 72 h, wound area (lower panel) was assessed by ImageJ and normalized to 0 h. c Boyden chamber migration assay was performed in H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells. Migrated cells were counted in 3 h. d Boyden chamber migration assay was performed in HuTu80/CD44 cells that were suspended in CM derived from H1299/Mock, H1299/SRGN or H1299/SRGN(S/A) cells, respectively. Migrated cells were counted in 5 h. e CM harvested from H1299/Mock and H1299/SRGN cells were treated with or without ChaseABC for 24 h, and subjected to western blot analysis of SRGN. HuTu80/CD44 cells were suspended in non-treated or ChaseABC-treated CM and subjected to Boyden chamber migration assay. Migrated cells were counted in 5 h. Data are presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by Student’s t -test

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Modification, Migration, Western Blot, Cell Culture, Wound Healing Assay, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: GAG modification is critical for SRGN binding to CD44. CM was harvested from H1299/Mock, H1299/SRGN or H1299/SRGN(S/A) cells, and incubated with CD44-Fc or IgG control for 18 h at 37 °C. Western blot analysis of SRGN in the input proteins and proteins bound to CD44-Fc and IgG using anti-SRGN antibody was shown

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Modification, Binding Assay, Incubation, Western Blot

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: SRGN induces cytoskeleton reorganization and Rho-family GTPase activation. a-c Phalloidin staining of actin. H1299/Mock and H1299/SRGN cells ( a ) were incubated in serum-free medium for 24 h and H460/sh-CTRL, and H460/sh-SRGN cells ( b ) were incubated in 2% FBS-containing medium for 72 h, followed by Alexa Fluor™ 594 Phalloidin staining of filamentous actin. c HuTu80/Mock and HuTu80/CD44 cells were seeded into the Culture-Insert 2 Well on slide and incubated overnight to form the gap for cell migration. After removing the insert, cells were washed with 1X PBS twice and incubated with CM harvested from H1299/SRGN cells that had been treated with or without ChaseABC for 24 h, followed by Alexa Fluor™ 594 Phalloidin staining of filamentous actin. Filopodia (arrows) and lamellipodia (arrowheads) structures were observed using confocal microscopy. The circumscribed rectangular regions are further enlarged for better viewing. d H1299/Mock, H1299/SRGN, H460/sh-CTRL, and H460/sh-SRGN cells were seeded in FN-coated dishes. In 30 min, attached cells were lysed and subjected for pulling down of Rac1-GTP, Cdc42-GTP and RhoA-GTP. Western blot analysis was performed for total and active Rac1, Cdc42, and RhoA as indicated

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Activation Assay, Staining, Incubation, Migration, Confocal Microscopy, Western Blot

Journal: Journal of Biomedical Science

Article Title: Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44

doi: 10.1186/s12929-019-0600-3

Figure Lengend Snippet: SRGN promotes cell migration through Src-elicited focal adhesion turnover. a After serum-starvation for 24 h, H1299/Mock and H1299/SRGN cells were plated in FN-coated dishes. In 30 min, cells were lysed and assayed for Src phosphorylation. In parallel, serum-starved cells were subjected to western blot analysis of phosphorylated paxillin and ERK/MAPK, and co-immunoprecipitation assay for paxillin/FAK adhesion complex formation. b H460/sh-CTRL and H460/sh-SRGN cells were cultured in serum-free medium for 24 h and examined for Src phosphorylation and paxillin/FAK complex formation. c H1299/SRGN cells were cultured in serum-free medium for 24 h, treated with PP2 (10 μM) or DMSO for 3 h, and phosphorylation of Src and paxillin, and paxillin/FAK complex formation were assessed. d Src expression was depleted in H1299/SRGN cells by shRNA-mediated knockdown approach, and phosphorylation of paxillin and the formation of paxillin/FAK complex were assessed in the control-knockdown (sh-CTRL) and Src-knockdown (sh-Src) cells. e-f H1299/Mock and H1299/SRGN cells (e) or H460/sh-CTRL and H460/sh-SRGN cells (f) were treated with or without PP2, and subjected to Boyden chamber migration assay. Migrated cells were counted in 3 h. Western blot analysis of phosphorylated Src and total Src was shown. Data are presented as the mean ± SD of three independent experiments. *** P < 0.001 by Student’s t-test. g HuTu80/CD44 cells were incubated with CM harvested from H1299/Mock and H1299/SRGN cells that had been treated with or without ChaseABC for 24 h, followed by western blot analysis of designated proteins. h H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells were cultured in serum-free medium for 24 h, and subjected to western blot analysis of phosphorylation of Src, paxillin and other proteins as indicated. i A working model is proposed for SRGN-mediated cell migration. SRGN in the tumor microenvironment binds to tumor cell surface CD44 via its CS-GAGs, and promotes Src activation and subsequent paxillin phosphorylation and the dissociation of paxillin/FAK adhesion complex, to accelerate focal adhesion turnover. SRGN also promotes the activation of Rac1 and Cdc42 to enhance cytoskeleton reorganization, leading to increased cell migration

Article Snippet: The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded.

Techniques: Migration, Western Blot, Co-Immunoprecipitation Assay, Cell Culture, Expressing, shRNA, Incubation, Activation Assay

Journal: Nature Communications

Article Title: Mucosal TLR5 activation controls healthspan and longevity

doi: 10.1038/s41467-023-44263-2

Figure Lengend Snippet: Kaplan-Meier survival graphs for female ( a , n = 25 biologically independent animals per group) and male ( b, n = 26 biologically independent animals per group) mice comparing vehicle control mice to those that received intranasally administered to FP fusion protein (6.5 μg) starting at 650 days of age. After the eight times of FP or vehicle administration, the aged mice were analyzed and compared to those of young mice. The results are displayed below: hair condition (c , O-Ctrl: n = 36; O-FP: n = 43 biologically independent animals), bone mineral density of the spine and femur determined by micro-CT ( d , Y: n = 13; O-Ctrl: n = 13; O-FP: n = 14 biologically independent animals), total area of tube formation of bone marrow-derived stem cells ( e , n = 10 biologically independent samples per group), weight of thymus ( f, Y: n = 35 samples; O-Ctrl: n = 36 samples; O-FP: n = 43 samples), number of thymocytes ( g , Y: n = 10 samples; O-Ctrl: n = 7 samples; O-FP: n = 12 samples), phenotypic analysis of naïve T cells (CD3 + CD44 lo CD62L hi ) in splenocytes ( h , Y: n = 10 samples; O-Ctrl: n = 7 samples; O-FP: n = 10 samples), micro-PET images of the mouse brains (left) and quantitative analysis of glucose uptake (right) ( i, Y: n = 12 biologically independent animals; O-Ctrl: n = 12 biologically independent animals; O-FP: n = 13 biologically independent animals), Cognitive functions were assessed with an open field test ( j , Y: n = 16 biologically independent animals; O-Ctrl: n = 8 biologically independent animals; O-FP: n = 16 biologically independent animals), the nest score ( k , Y: n = 9 biologically independent animals; O-Ctrl: n = 9 biologically independent animals; O-FP: n = 8 biologically independent animals), the preference score ( l, Y: n = 10 biologically independent animals; O-Ctrl: n = 7 biologically independent animals; O-FP: n = 8 biologically independent animals), and the passive avoidance test ( m , Y: n = 7 biologically independent animals; O-Ctrl: n = 6 biologically independent animals; O-FP: n = 6 biologically independent animals). Error bars represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 using the Log - rank (Mantel-Cox) test ( a and b ), the unpaired two-tailed t test ( c ), and the one-way ANOVA ( d–m ). ns, not significant; FP, FlaB-PspA fusion proteins; Ctrl, vehicle control group of aged mice; Y, young mice (8 weeks old); SUV, standardized uptake value. Source data are provided as a Source Data file.

Article Snippet: All of the following antibodies for FACS were purchased from BD Biosciences or Novus Biologicals: CD3-FITC, CD44-PE-Cy7, CD62L-APC, TLR5-FITC (Novus Biologicals, Centennial CO, USA).

Techniques: Micro-CT, Derivative Assay, Micro-PET, Two Tailed Test

Journal: Molecular Cancer

Article Title: DCLK1 drives malignant progression and chemoresistance of bladder cancer by deubiquitinating HDAC6

doi: 10.1186/s12943-025-02560-y

Figure Lengend Snippet: DCLK1 knockdown suppresses stemness and enhances chemosensitivity in bladder cancer cells. A mRNA expression levels of DCLK1 in bladder cancer tissues compared with normal tissues. B qRT-PCR analysis of DCLK1 expression in BC cell lines and SV-HUC-1 cells. C qRT-PCR analysis confirming the knockdown efficiency of DCLK1. D Representative images of tumorsphere formation in DCLK1-depleted BC cells (scale bar = 100 μm). E Western blot analysis of stemness-associated markers (ALDH1, CD133, CD44, Nanog, and SOX2) in DCLK1-silenced BC cells. F The proportions of ALDH1⁺ and CD44⁺ cells were significantly higher in the control group than in the DCLK1-knockdown group under cancer stem cell-enriched conditions. G Cisplatin-induced apoptosis in BC cells analyzed by Annexin V/PI staining followed by flow cytometry. H Cisplatin-induced apoptosis in BC cells examined by the TUNEL assay (scale bar = 20 μm). Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001

Article Snippet: DCLK1 (1:1000; Cell Signaling, 62257), HDAC6 (1:1000; Cell Signaling, 7558), USP10 (1:1000; Proteintech, 8501), E-cadherin (1:1000; Cell Signaling, 3195), Vimentin (1:1000; Cell Signaling, 5741), β-catenin (1:1000; Cell Signaling, 9562), Flag (1:1000; Sigma, F1804), HA (1:1000; Sigma, H6908), Myc-tag (1:1000; Proteintech, 16286-1-AP), α-tubulin (1:1000; Proteintech, 66031-1-Ig), ALDH1 (1:1000; Cell Signaling, 54135), CD133 (1:1000; Cell Signaling, 64326), CD44 (1:1000; Cell Signaling, 37259),SOX2 (1:1000; Cell Signaling, 23064),Nanog (1:2000; Cell Signaling, 4903), Notch1 (1:1000; Cell Signaling, 3608), c-Myc (1:1000; Cell Signaling, 13987), MAML1 (1:1000; Cell Signaling, 12166), HES1 (1:1000; Cell Signaling, 11988), HEY1 (1:1000; abcam, 154077), PD-L1 (1:1000; Cell Signaling, 13684), β-Actin (1:10000; Cell Signaling, 4967) and GAPDH (1:5000; Abcam, ab128915).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Control, Staining, Flow Cytometry, TUNEL Assay