Journal: Journal of mammary gland biology and neoplasia

Article Title: FGFR2 Controls Growth, Adhesion and Migration of Nontumorigenic Human Mammary Epithelial Cells by Regulation of Integrin β1 Degradation.

doi: 10.1007/s10911-023-09537-x

Figure Lengend Snippet: Fig. 3 FGFR2 regulates degradation of integrin β1. (a) Relative expression of ITGB1 in HB2 and HB2 FGFR2(-) cells was analysed by RT-qPCR. Data presented as means ± SD (n = 3). (b) HB2 cells were incubated with Leupeptin (100 µg/ml) and MG132 (200 nM) for 24, 48 and 72 h. Integrin β1 protein level was analysed by Western blotting. β-actin was used as a protein loading control. (c) HB2 FGFR2(-) cells were incubated with Leupeptin (100 µg/ml) and MG132 (200 nM) for 24, 48 and 72 h. Integrin β1 protein level was analysed by Western blot ting. β-actin was used as a protein loading control

Article Snippet: The following antibodies were used in this study: β-actin (Sigma-Aldrich, clone AC-74), FGFR1 (Santa Cruz Biotechnology, sc-121), FGFR2 (Cell Signaling, #23328), integrin β1 (Cell Signaling, #4706; Santa Cruz Biotechnology, sc-9970), integrin α1 (Santa Cruz Biotechnology, sc-10728), integrin α2 (Santa Cruz Biotechnology, sc-9089), integrin α3 (Santa Cruz Biotechnology, sc-6592), integrin α5 (Chemicon International, AB1949), integrin α6 (Cell Signaling, #3750), LAMP1 (R&D Systems, MAB4800), FGFR (Tyr653/654) (Cell Signaling, #3471), ERK1/2 (Thr202/ Tyr204) (Cell Signaling, #9101), DyLightTM 488-conjugated AffiniPure Goat Anti-Rabbit (Jackson ImmunoResearch), AlexaFluor® 680-conjugated AffiniPure Goat Anti-Rabbit (Jackson ImmunoResearch), DyLightTM 594-conjugated AffiniPure Donkey Anti-Mouse (Jackson ImmunoResearch), AlexaFluor® 790-conjugated AffiniPure Donkey Anti-Mouse (Jackson ImmunoResearch), Mouse Anti-Goat IgG-HRP (Santa Cruz Biotechnology, sc-2354).

Techniques: Expressing, Quantitative RT-PCR, Incubation, Western Blot, Control

Journal: Oncoimmunology

Article Title: IRE1α overexpression in malignant cells limits tumor progression by inducing an anti-cancer immune response

doi: 10.1080/2162402X.2022.2116844

Figure Lengend Snippet:

Article Snippet: CD3 + cells were depleted by magnetic isolation using an autoMACS Pro Separator (Miltenyi Biotec) after staining with FITC-conjugated antibodies against CD19 (Miltenyi, #130-102-494), CD45R (Miltenyi, #130-110-708), CD49b (Miltenyi, #130-102-258), CD11b (BD Bioscience, #553310) and Ter-119 (Miltenyi, #130-112-719).

Techniques:

Journal: Journal of Skin Cancer

Article Title: Protein Kinase C ε , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells

doi: 10.1155/2013/452425

Figure Lengend Snippet: Hair follicle stem cells and clonogenicity of epidermal keratinocytes isolated from PKC ε overexpressing mice and wild-type littermates. (a) is showing the representative gating of epidermal stem cell population in a dot plot from untreated WT, TG224, and TG215 mice. In each dot plot, the upper right quadrant is representing the CD34+/ α 6-integrin+ (double positive HSCs) stem cell population. Each value in the histogram is an average of FACS analysis of triplicate samples from keratinocytes pooled from two mice. (b) represents the total frequency of CD34+/ α 6-integrin+ keratinocytes in untreated indicated mice. (c) and (d) Clonogenicity of epidermal keratinocytes. Briefly, the keratinocytes from 7-8 weeks old indicated that mice were harvested using SMEM harvesting medium. Irradiated 3T3 cells seeded at density 10 6 cells/dish to the 60 mm dishes a day before seeding keratinocytes. For feeder layer, irradiated 3T3 cells were cultured in EMEM medium with 10% FBS and 1% penicillin-streptomycin. Equal numbers of keratinocyte cells (3000 cells/dish) were seeded for each type of mice and cultured with William's E media for 2 weeks. For counting and measurement of colonies, dishes were fixed with 10% formalin and stained with 0.5% rhodamine B. (c) Keratinocyte colonies. Shown are the representative dishes of adult keratinocyte colonies from PKC ε TG mice and their WT littermates. (d) Quantitation of colonies. The colonies were counted and colony size measured by using vernier caliper . Each value is the mean ±SE of colonies from 4–7 dishes.

Article Snippet: Freshly harvested keratinocytes were incubated with PE-conjugated anti- α 6-integrin and FITC-conjugated anti-CD34 antibodies, fixed and stained using the APC BrdU Flow Kit, following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA).

Techniques: Isolation, Irradiation, Cell Culture, Staining, Quantitation Assay

Journal: Journal of Skin Cancer

Article Title: Protein Kinase C ε , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells

doi: 10.1155/2013/452425

Figure Lengend Snippet: Effects of single UV exposure on live epidermal stem cell population determined by flow cytometric analysis. PKC ε overexpressing TG and their WT littermates were exposed once to UV (1.8 kJ/m 2 ). At the indicated times after UV, mice were sacrificed and the dorsal skin removed for keratinocyte isolation as previously described . (a) and (c) Percent distribution of FACS-sorted keratinocytes following UV exposure of the indicated mice at the indicated times after UV exposure. (b) and (d) Frequency of total double positive HSCs (CD34+/ α 6-integrin+) in TG224, TG215, and their WT littermates at the indicated times after single UV exposure.

Article Snippet: Freshly harvested keratinocytes were incubated with PE-conjugated anti- α 6-integrin and FITC-conjugated anti-CD34 antibodies, fixed and stained using the APC BrdU Flow Kit, following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA).

Techniques: Isolation

Journal: Journal of Skin Cancer

Article Title: Protein Kinase C ε , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells

doi: 10.1155/2013/452425

Figure Lengend Snippet: Effects of chronic UV exposures on live epidermal stem cell population determined by flow cytometric analysis. PKC ε overexpressing TG and their WT littermates were given total eight UV exposures (1.8 kJ/m 2 , Monday, Wednesday, and Friday). At 24 hr after the last UV exposure, mice were sacrificed and the dorsal skin removed for keratinocyte isolation. (a) Percent distribution of FACS-sorted keratinocytes following UV exposures of the indicated mice. (b) Frequency of total double positive HSCs (CD34+/ α 6-integrin+) keratinocytes in UV-treated mice.

Article Snippet: Freshly harvested keratinocytes were incubated with PE-conjugated anti- α 6-integrin and FITC-conjugated anti-CD34 antibodies, fixed and stained using the APC BrdU Flow Kit, following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA).

Techniques: Isolation

Journal: Journal of Skin Cancer

Article Title: Protein Kinase C ε , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells

doi: 10.1155/2013/452425

Figure Lengend Snippet: PKC ε overexpressing transgenic mice have increased turnover of HSCs as determined by BrdU retaining double positive HSCs (CD34+/ α 6-integrin+). To identify LRCs, newborn mice (3 days old) were injected subcutaneously with BrdU (50 mg/Kg body weight) twice daily for 3 days. Mice were then sacrificed at 8 weeks after the last BrdU injection. There were four mice per group. (a) Representative gating of cell populations for side scatter (SSC) versus forward scatter (FSC). (b) Representative gating of cell populations at 5 and 8 weeks in the indicated mice. (c) Frequency of total BrdU-labeled double positive (CD34+/ α 6-integrin+) keratinocytes in TG 224 and their WT littermates at 4, 5, and 8 weeks.

Article Snippet: Freshly harvested keratinocytes were incubated with PE-conjugated anti- α 6-integrin and FITC-conjugated anti-CD34 antibodies, fixed and stained using the APC BrdU Flow Kit, following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA).

Techniques: Transgenic Assay, Injection, Labeling

Journal: Journal of Skin Cancer

Article Title: Protein Kinase C ε , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells

doi: 10.1155/2013/452425

Figure Lengend Snippet: Distribution and expression of PKC ε in FACS-sorted keratinocytes from WT, TG224, and TG215 mice. (a) FACS-sorted keratinocytes. The keratinocytes were harvested from 5 weeks old wild-type and TG mice and incubated with the CD34 and α 6-integrin florescent antibodies. After labeling, the cells were washed twice, filtered, and sorted for CD34+, α 6-integrin+ cells, CD34+/ α 6-integrin+, and CD34−/ α 6-integrin-cells. (b) PKC ε expression. The RNA was isolated from the sorted cell, followed by cDNA preparation, and then real-time PCR using SYBR Green double-strand DNA binding dye. After real-time PCR Ct values were calculated and analyzed for expression. All the expression values shown in the figures are relative to their mouse Gapdh internal control.

Article Snippet: Freshly harvested keratinocytes were incubated with PE-conjugated anti- α 6-integrin and FITC-conjugated anti-CD34 antibodies, fixed and stained using the APC BrdU Flow Kit, following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Incubation, Labeling, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Binding Assay

Journal: Journal of Skin Cancer

Article Title: Protein Kinase C ε , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells

doi: 10.1155/2013/452425

Figure Lengend Snippet: List of differentially expressed genes in CD34+/ α 6-integrin+ stem cells from PKC ε TG mice. Overexpression of PKC ε in the epidermis results in increased expression of genes linked to cell transformation, invasion, and metastasis.

Article Snippet: Freshly harvested keratinocytes were incubated with PE-conjugated anti- α 6-integrin and FITC-conjugated anti-CD34 antibodies, fixed and stained using the APC BrdU Flow Kit, following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA).

Techniques: Over Expression, Expressing, Transformation Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: (A) Expression of integrin α6 in papillary lysates from a 3-day old Itga6flox/flox and Hoxb7cre:Itga6flox/flox mice was analyzed by Western blotting analysis. α6 is indicated by the arrowhead. (B–I) Morphology of H&E stained kidney sections from Itga6flox/flox and Hoxb7cre:Itga6flox/flox mice at 2 months (magnification 40×, scale bar =500um) (B, C), 6 months (magnification 40×, scale bar =500um), (D, E) and 10 months of age (F, G) (magnification 40×, scale bar =500um) and (H, I) (magnification 200×, scale bar =100um).

Article Snippet: Anti-mouse β1 (550530), α1 (555001), α2 (553819), α5 (553350), α6 (555734) and αv (550024) integrin antibodies were purchased from BD Biosciences.

Techniques: Expressing, Western Blot, Staining

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: A) Expression of integrin β4 in papillary lysates from a 3-day old Itgb4flox/flox and Hoxb7cre:Itgb4flox/flox mice analyzed by Western blotting analysis. (B–I) Morphology of H&E stained kidney sections from Itgb4flox/flox and Hoxb7cre:Itgb4flox/flox mice at 2 months (magnification 40×, scale bar =500um) (B, C), 6 months (magnification 40×, scale bar =500um), (D, E) and 12 months of age (F, G) (magnification 40×, scale bar =500um) and (H, I) (magnification 200×, scale bar =100um).

Article Snippet: Anti-mouse β1 (550530), α1 (555001), α2 (553819), α5 (553350), α6 (555734) and αv (550024) integrin antibodies were purchased from BD Biosciences.

Techniques: Expressing, Western Blot, Staining

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: (A) Surface expression of integrin α6, β1 and β4 subunits were determined on Itgα6f/f and Itgα6−/− CD cells by flow cytometry using R-phycoerythrin (PE) conjugated secondary antibodies. (B) Lysates from Itgα6f/f and Itgα6−/− CD cells (20 μg total protein/lane) were immunoblotted for integrin α3 subunits or β-actin (loading control). Adhesion (C), migration (D) and proliferation (E) on LM-511 were evaluated as described in the Methods. For migration and proliferation, 1 μg/ml LM-511 was used. Shown are mean measurements ±SEM of 4–6 independent experiments. (F) Itgα6f/f and Itgα6−/− CD cells were plated in serum-free medium on LM-511 (1 μg/ml). Cells were lysed at 30, 60 and 90 min after plating and lysates (20 μg total protein/lane) were analyzed by Western blot for levels of phosphorylated and total Akt, p38, and ERK1/2 or β-actin (loading control).

Article Snippet: Anti-mouse β1 (550530), α1 (555001), α2 (553819), α5 (553350), α6 (555734) and αv (550024) integrin antibodies were purchased from BD Biosciences.

Techniques: Expressing, Flow Cytometry, Migration, Western Blot

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Integrin alpha6 maintains the structural integrity of the kidney collecting system

doi: 10.1016/j.matbio.2016.12.003

Figure Lengend Snippet: Adhesion (A), migration (B), proliferation (C), and replating assay (D) on LM-322 (1 μg/ml) were evaluated as described in the Methods and in Fig. 5. Shown are mean measurements ±SEM of 4–6 independent experiments; *p<0.01between Itgα6−/− and Itgα6f/f CD cells. (E) The shear stress for 50% detachment (τ50), which represents the mean cell adhesion strength, was determined for Itgα6−/− and Itgα6f/f CD cells on LM-332 and fibronectin (which is not a ligand of a6 containing integrin) coated cell culture plates. Shown are the averaged τ50 values of 3 independent experiments; *p<0.01between Itgα6−/− and Itgα6f/f CD cells.

Article Snippet: Anti-mouse β1 (550530), α1 (555001), α2 (553819), α5 (553350), α6 (555734) and αv (550024) integrin antibodies were purchased from BD Biosciences.

Techniques: Migration, Cell Culture