Journal: International Journal of Molecular Sciences

Article Title: Mitigation of Breast Cancer Cells’ Invasiveness via Down Regulation of ETV7, Hippo, and PI3K/mTOR Pathways by Vitamin D3 Gold-Nanoparticles

doi: 10.3390/ijms25105348

Figure Lengend Snippet: ( A ) Representative Western blots of PI3K, AKT, p-AKT(Ser473), mTOR, and ETV7 in control ( C ) and VD3-GNP-treated MDA-MB-231 cells. * p < 0.05. ( B ) Calculation of the protein expression (band density) in Western blot analysis (n = 3) in MDA-MB-231 cells. Protein levels of PI3K, AKT, p-AKT (Ser473), mTOR, and ETV7 were normalized by β-actin, * p < 0.05, ** p < 0.001. ( C ) representative Western blots of PI3K, AKT, p-AKT (Ser473), mTOR, and ETV7 in control ( C ), and VD3-GNP-treated MCF-7 cells. β-actin was used as a loading control. ( D ) The protein expression (band density) in Western blot analysis (n = 3) for protein levels in MCF-7 cells was normalized by β-actin, * p < 0.05, ** p < 0.001.

Article Snippet: PVDF membranes were blocked with 5% BSA in TBS-T at room temperature for 1 h and were subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with anti-mouse IgG (1:10,000, Jackson-Immuno Research, West Grove, PA, USA, 115035068) in TBS-T for 1 h. Primary antibodies for PI3K (Developmental Studies Hybridoma Bank (DSHB), AFFN-PIK3R2-3D4), mTOR (DSHB, CPTC-MTOR-1), AKT (DSHB, CPTC-AKT1-1), ETV7 (DSHB, PCRP-ETV7-1A1), YAP (DSHB, YAP2), Taz (DSHB, PCRP-ZBTB18-1B2), p-AKT (Ser473), and p-YAP (Ser127), (Cell Signaling Technology, Danvers, MA, USA, 9271) were used (1:100 in 0.5% TBS-T) and β-actin antibody conjugated to peroxidase was used as a loading control (Sigma-Aldrich, A3854, dilution 1:50,000).

Techniques: Western Blot, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Mitigation of Breast Cancer Cells’ Invasiveness via Down Regulation of ETV7, Hippo, and PI3K/mTOR Pathways by Vitamin D3 Gold-Nanoparticles

doi: 10.3390/ijms25105348

Figure Lengend Snippet: PPIs (using the STRING database) for the PI3K/mTOR/AKT (Gene IDs: PIK3C, MTOR, AKT1, ETV7) and Hippo pathways (Gene IDs: Yap1, TAZ), with 10 functional proteins. ( A ) AKT1; ( B ) MTOR; ( C ) PI3K; ( D ) ETV7; ( E ) YAP; ( F ) TAZ.

Article Snippet: PVDF membranes were blocked with 5% BSA in TBS-T at room temperature for 1 h and were subsequently incubated with primary antibodies overnight at 4 °C, followed by incubation with anti-mouse IgG (1:10,000, Jackson-Immuno Research, West Grove, PA, USA, 115035068) in TBS-T for 1 h. Primary antibodies for PI3K (Developmental Studies Hybridoma Bank (DSHB), AFFN-PIK3R2-3D4), mTOR (DSHB, CPTC-MTOR-1), AKT (DSHB, CPTC-AKT1-1), ETV7 (DSHB, PCRP-ETV7-1A1), YAP (DSHB, YAP2), Taz (DSHB, PCRP-ZBTB18-1B2), p-AKT (Ser473), and p-YAP (Ser127), (Cell Signaling Technology, Danvers, MA, USA, 9271) were used (1:100 in 0.5% TBS-T) and β-actin antibody conjugated to peroxidase was used as a loading control (Sigma-Aldrich, A3854, dilution 1:50,000).

Techniques: Functional Assay

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: Signaling Molecules of Human Skin Cells as the Targets for Injection Cosmetology

doi: 10.2147/CCID.S321104

Figure Lengend Snippet: Expression of melatonin receptor 1A in keratinocytes enriched with Merkel cell culture at passage 1 and 3; DAPI stained nuclei; expression of cytokeratin-20 – green fluorescence; expression of the protein under study – red fluorescence; magnification x40.

Article Snippet: The following primary monoclonal antibodies were used for an immunocytochemical investigation: type I collagen (1:150, Abcam), elastin (1:50, Abcam), AP-1 (1:200, Sigma), Klotho (1:250, Abcam), MTH-1 (1:1000, Abcam), melatonin receptor 1A (1:200, Abcam), melatonin receptor 1B (1:100, Abcam), clock (1:100, Abcam), cytokeratin 20 (1:150, Dako).

Techniques: Expressing, Cell Culture, Staining, Fluorescence

Journal: Stem Cells International

Article Title: Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching

doi: 10.1155/2016/7342379

Figure Lengend Snippet: (a) Microphotographs of LIVE/DEAD® staining of seeded scaffold samples (viable cells shown in green, dead cells in red; left), hematoxylin and eosin staining of paraffin sections (middle), and procollagen 3A1 immunostaining of paraffin sections (positive staining in dark brown; right), 24 h after mechanical stimulation. (b) Score points for cell viability (left) or cell morphology, alignment, and distribution on the scaffold surface (L/D score; middle), obtained by evaluation of LIVE/DEAD stained samples, the horizontal lines representing the monolayer controls, and score points for cell integration and cell layer integrity (HE score, right), obtained by evaluation of hematoxylin and eosin stained sections; bars represent the median values and error bars the 95% confidence interval; ⋆ marks significant differences compared to the monolayer control ( p < 0.05); # means significant differences between the sample groups indicated ( p < 0.05); stat: static; stim: mechanical stimulation.

Article Snippet: Immunostaining was performed using goat anti-human procollagen 1A1 polyclonal antibodies or mouse anti-human procollagen 3A1 monoclonal antibodies (A-17 or B-4; Santa Cruz Biotechnology, Heidelberg, Germany) combined with Immunocruz TM ABC staining systems (Santa Cruz Biotechnology) containing donkey anti-goat or goat anti-mouse secondary antibodies.

Techniques: Staining, Immunostaining, Control

Journal: Journal of Biological Chemistry

Article Title: Up-regulation of Cholesterol Absorption Is a Mechanism for Cholecystokinin-induced Hypercholesterolemia

doi: 10.1074/jbc.m113.534388

Figure Lengend Snippet: FIGURE 3. CCK induces cholesterol absorption in Caco-2 cells by regula- tion of NPC1L1 protein membrane translocation. A, Caco-2 cells grown in transwell inserts were treated with 10 nM [Thr28,Nle31]CCK in the basolateral compartment for the indicated time periods. NPC1L1 mRNAs were deter- mined with real-time RT-PCR and quantitated relative to the GAPDH mRNA level.BandC,Caco-2cellsgrownintranswellinsertsweretreatedwithculture medium alone as a control (ctrl) or 10 nM [Thr28,Nle31]CCK in the basolateral compartment for 30 or 120 min. D and E, Caco-2 cells were transfected with scrambled (Scrmbl) siRNA or siRNA specific for NPC1L1 and then treated with culturemediumaloneasacontrolor10nM[Thr28,Nle31]CCKinthebasolateral compartment for 30 min. Apical membrane proteins were isolated by biotin precipitation. NPC1L1 (1L1), SI, and CYP1a1 (1a1) proteins in the membrane and the total cell lysate were determined with Western blot analysis. The levels of membrane (Mbrn) and total NPC1L1 were expressed relative to the total level of -actin. F, Caco-2 cells transfected with scrambled siRNA or siRNA specific for NPC1L1 were incubated with 0.002 Ci of [3H]cholesterol micelles in the apical compartment and with culture medium alone (control) or 10 nM [Thr28,Nle31]CCK in the basolateral compartment. Absorbed [3H]cho- lesterol was determined by radioactivity counting of the basolateral medium. Values represent the mean S.E. (error bars) of five independent experi- ments. *, p 0.05 versus cells incubated with medium alone (control); †, p 0.05 versus cells with scrambled siRNA transfection and without CCK treat- ment; ‡, p 0.05 versus cells with scrambled siRNA transfection and CCK treatment.

Article Snippet: Protein A/G Plusagarose, Akt inhibitor XI (sc221229), scrambled (sc37007) and NPC1L1 siRNA (sc61225); Rab11a siRNAs (sc36340); PI3K p85 siRNA (sc36217); horseradish peroxidase- and fluorescein isothiocyanate-conjugated second antibodies (sc2314, sc2020, and sc2010); and primary antibodies against human NPC1L1 (sc166802), Rab11a (sc58465), phosphorylated Akt (sc7985-R), cytochrome P450 1a1 (CYP1a1) (sc9828), sucrase isomaltase (SI) (sc-393424), and CCK1R (sc43670) were purchased from Santa Cruz Biotechnology.

Techniques: Membrane, Translocation Assay, Quantitative RT-PCR, Control, Transfection, Isolation, Western Blot, Incubation, Radioactivity