Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy

doi: 10.1158/1078-0432.CCR-17-3435

Figure Lengend Snippet: (A-C) Panc-1 and MIA PaCa-2 were transfected with scrambled siRNA or SMAD4-specific siRNA for 48 h and then were treated with or without IR (6 Gy). Cell lysates were harvested at the indicated time points after IR. (A) Immunoblot of LC3 isoforms were measured using antibody against LC3. Quantification of the LC3-II/LC3-I ratio in Panc-1 cells (B) and MIA PaCa-2 cells (C). The protein level of the β-actin was used as the internal standard. Shown are the averages of triplicate experiments. (D) Representative confocal images of endogenous LC3 staining in Panc-1 transfected with scramble or SMAD4-specific siRNA for 48 h, and treated with or without IR (6 Gy). Cells were stained with antibody to LC3 (red) and DAPI (blue). Scale bars, 10 μm. (E) Quantification of LC3 puncta in Panc-1 cell lines.

Article Snippet: After quenching endogenous peroxidase activity with H 2 O 2 and blocking with 10% horse serum, the sections were incubated sequentially with the primary antibodies mouse anti-SMAD4 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-LC3 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-γ-H2AX (1:100; Cell Signaling Technology Inc. CA, USA), biotinylated secondary antibodies, and the Avidin-Biotin Complexes (ABC) reagent (Gene Tech.

Techniques: Transfection, Western Blot, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy

doi: 10.1158/1078-0432.CCR-17-3435

Figure Lengend Snippet: (A) Schemes for the establishment and treatment of the pancreatic cancer orthotopic model. (B, C) Nude mice were injected with 1×106 Panc-1 cells infected with control retroviral vector, or shSMAD4 vector with 5% Matrigel into the pancreas. After 21 days, tumors were irradiated thrice with 4-Gy dose of IR every other day. The mice were sacrificed at day 42, and the tumors were isolated, weighed, and compared between the groups using ANOVA. (D) Immunohistochemistry (IHC) was performed on serial sections using antibodies against SMAD4, LC3 and g-H2AX in tumor tissues.

Article Snippet: After quenching endogenous peroxidase activity with H 2 O 2 and blocking with 10% horse serum, the sections were incubated sequentially with the primary antibodies mouse anti-SMAD4 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-LC3 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-γ-H2AX (1:100; Cell Signaling Technology Inc. CA, USA), biotinylated secondary antibodies, and the Avidin-Biotin Complexes (ABC) reagent (Gene Tech.

Techniques: Injection, Infection, Plasmid Preparation, Irradiation, Isolation, Immunohistochemistry

Journal: PLoS ONE

Article Title: Long-Term Treatment with the Sodium Glucose Cotransporter 2 Inhibitor, Dapagliflozin, Ameliorates Glucose Homeostasis and Diabetic Nephropathy in db/db Mice

doi: 10.1371/journal.pone.0100777

Figure Lengend Snippet: (A, B) ROS production was detected by fluorescence microscopy using dihydroethidium (DHE) staining. ROS was predominantly localized in the interstitia of db/db mice, and was suppressed in the db/db +dapa groups compared with that in the db/db group. Original magnification, ×100. Data are mean ± SEM. * P <0.05. (C, D) Localization of Nox4 was detected by immunohistochemistry. The expression of Nox4 was predominantly localized in the interstitia of db/db mice, and was suppressed in the db/db +dapa groups compared with that in in the db/db group. Original magnification, ×100. Data are mean ± SEM. * P <0.05.

Article Snippet: Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore).

Techniques: Fluorescence, Microscopy, Staining, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Long-Term Treatment with the Sodium Glucose Cotransporter 2 Inhibitor, Dapagliflozin, Ameliorates Glucose Homeostasis and Diabetic Nephropathy in db/db Mice

doi: 10.1371/journal.pone.0100777

Figure Lengend Snippet: (A) ROS production was detected by fluorescence microscopy using dihydroethidium (DHE) staining. ROS production was not increased by mannitol (b) compared with normal glucose (a), but was increased by high glucose (c). High-glucose-induced ROS production was decreased by dapagliflozin pretreatment in a dose-dependent manner (d: 0.2 nM; e: 2.0 nM; f: 20.0 nM). (B) Densitometric quantification of ROS production. Data are mean ± SEM. * P <0.05 vs. high glucose; NG: normal glucose; Man: mannitol; HG: high glucose; dapa: dapagliflozin. Quantitative RT-PCR analysis of the expression of Nox4 (C), MCP-1 (D) and osteopontin (E) showed that dapagliflozin inhibited diabetes-induced inflammation in the kidney. mRNA levels were normalized against Atp5f1 expression. Data are mean ± SEM. * P <0.05.

Article Snippet: Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore).

Techniques: Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: Fluorescein punctate staining are the fluorescein-incorporated corneal epithelial cells of the superficial layer featured with decreased MUC1 expression. (A) The clinical or slit lamp view under cobalt blue light of the eyes of sham-operated rabbit model (lower panel) or dry eye rabbit model ( upper panel ) following fluorescein staining. (B) The fluorescein-stained corneal epithelia prepared as the frozen tissue sections were stained with MUC1 for immunohistochemistry ( brown , left panel ) and immunofluorescence ( middle and right panel ) analyses. For immunofluorescence, dual channels ( red and blue , middle panel ) or triple channels ( red , blue , and green , right panel ) were used. The cell nuclei were counterstained with hematoxylin ( purple , immunohistochemistry) or DAPI ( blue , immunofluorescence). (C) The frozen corneal epithelia section prepared from sham-operated and lacrimal gland-removed rabbits were immunostained with ZO-1 ( red ), nuclear counterstained with DAPI ( blue ), and the fluorescein ingress ( green ) were analyzed through a confocal microscopy. ( red and blue dual channels, middle panel ; red , blue , and green triple channels, right panel ).

Article Snippet: The cryosections were then stained with a biotin-conjugated anti-rabbit MUC1 rabbit antibody (no. NBP1-60046B; Novus Biologicals, Littleton, CO, USA), anti-ZO-1 (no. 33-9100; ThermoFisher Scientific, Waltham, MA, USA), or anti-galectin-3 (GAL3) mouse antibody (no. A3A12, Novus Biologicals) for one hour followed by incubation in a Texas Red conjugate of NeutrAvidin biotin-binding protein (no. A2665; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 594 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (no. A-11012; Invitrogen), or Alexa Fluor 564-conjugated goat anti-mouse IgG antibody (no. A-11003) for one hour in the darkness.

Techniques: Staining, Expressing, Immunohistochemistry, Immunofluorescence, Confocal Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: The fluorescein intensity is reversely correlated with the level of MUC1 in HCECs cultured in MPM. (A) The HCECs that have been grown in KSFM were cultured in MPM for two or five days, and the levels of MUC1, MUC4, and MUC16 mRNA were examined. (B) Representative flow cytometric analyses of MUC1 expressions on cell membranes of HCECs cultured in KSFM ( left ) or MPM ( right ) were presented in contour plots. (C) Representative flow cytometric analyses of fluorescence intensity and MUC1 expression in HCECs cultured in KSFM ( left ) or MPM ( right ) followed by fluorescein staining were presented in contour plots. (D) The HCECs cultured in KSFM or MPM were stained with fluorescein, and the levels of fluorescence ( green ) and MUC1 ( red ) were examined using an immunofluorescence microscopy. Nuclei were stained with DAPI ( blue ). The images were obtained via a confocal microscopy.

Article Snippet: The cryosections were then stained with a biotin-conjugated anti-rabbit MUC1 rabbit antibody (no. NBP1-60046B; Novus Biologicals, Littleton, CO, USA), anti-ZO-1 (no. 33-9100; ThermoFisher Scientific, Waltham, MA, USA), or anti-galectin-3 (GAL3) mouse antibody (no. A3A12, Novus Biologicals) for one hour followed by incubation in a Texas Red conjugate of NeutrAvidin biotin-binding protein (no. A2665; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 594 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (no. A-11012; Invitrogen), or Alexa Fluor 564-conjugated goat anti-mouse IgG antibody (no. A-11003) for one hour in the darkness.

Techniques: Cell Culture, Fluorescence, Expressing, Staining, Immunofluorescence, Microscopy, Confocal Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: Diquafosol treatments preferentially promotes the expression of MUC1 on cell membranes. (A) The HCECs were grown in a culture media containing 0, 0.5, or 1.0 mM diquafosol for four days, and the levels of MUC1, MUC4, and MUC16 mRNA of treated cells were examined using qRT-PCR. (B) Representative flow cytometric analyses of MUC1, MUC4, and MUC16 expression on cell membranes of HCECs treated with 1 mM diquafosol ( red ) or control ( gray ) were presented in overlaid histograms of signal intensity. (C) Representative flow cytometric analyses of fluorescence intensity and MUC1 expression in HCECs treated with sham ( left ) or 1 mM diquafosol ( right ) followed by fluorescein staining were presented in contour plots.

Article Snippet: The cryosections were then stained with a biotin-conjugated anti-rabbit MUC1 rabbit antibody (no. NBP1-60046B; Novus Biologicals, Littleton, CO, USA), anti-ZO-1 (no. 33-9100; ThermoFisher Scientific, Waltham, MA, USA), or anti-galectin-3 (GAL3) mouse antibody (no. A3A12, Novus Biologicals) for one hour followed by incubation in a Texas Red conjugate of NeutrAvidin biotin-binding protein (no. A2665; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 594 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (no. A-11012; Invitrogen), or Alexa Fluor 564-conjugated goat anti-mouse IgG antibody (no. A-11003) for one hour in the darkness.

Techniques: Expressing, Quantitative RT-PCR, Control, Fluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: Knockdown of MUC1 leads to an increase in fluorescein ingress. (A) The mRNA levels of MUC1, MUC4, and MUC16 in HCECs separately transduced with two lentiviral-based shRNA vectors (shMUC1-1 or shMUC1-2) were examined using RT-PCR. The β-actin gene was used as an internal control. (B) Representative flow cytometric analyses of fluorescence intensity and MUC1 expressions in transduced HCECs followed by fluorescein staining were shown in contour plots.

Article Snippet: The cryosections were then stained with a biotin-conjugated anti-rabbit MUC1 rabbit antibody (no. NBP1-60046B; Novus Biologicals, Littleton, CO, USA), anti-ZO-1 (no. 33-9100; ThermoFisher Scientific, Waltham, MA, USA), or anti-galectin-3 (GAL3) mouse antibody (no. A3A12, Novus Biologicals) for one hour followed by incubation in a Texas Red conjugate of NeutrAvidin biotin-binding protein (no. A2665; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 594 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (no. A-11012; Invitrogen), or Alexa Fluor 564-conjugated goat anti-mouse IgG antibody (no. A-11003) for one hour in the darkness.

Techniques: Knockdown, Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Control, Fluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: The overexpression of MUC1 lacking the extracellular domain is unable to block fluorescein ingress. (A) Flow cytometric analyses of keratin 3, keratin 12, and vimentin expressions in HCECs cultured in KSFM ( gray ) or MPM ( red ) were presented in overlaid histograms of signal intensity. (B) Schematic diagrams of the wild-type and N-terminally truncated MUC1. (C) Representative flow cytometric analyses of fluorescence intensity and MUC1-C levels in HCECs transfected with the control vector ( left ) or the vector encoded with N-terminally truncated MUC1s ( right ) followed by fluorescein staining were shown in contour plots.

Article Snippet: The cryosections were then stained with a biotin-conjugated anti-rabbit MUC1 rabbit antibody (no. NBP1-60046B; Novus Biologicals, Littleton, CO, USA), anti-ZO-1 (no. 33-9100; ThermoFisher Scientific, Waltham, MA, USA), or anti-galectin-3 (GAL3) mouse antibody (no. A3A12, Novus Biologicals) for one hour followed by incubation in a Texas Red conjugate of NeutrAvidin biotin-binding protein (no. A2665; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 594 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (no. A-11012; Invitrogen), or Alexa Fluor 564-conjugated goat anti-mouse IgG antibody (no. A-11003) for one hour in the darkness.

Techniques: Over Expression, Blocking Assay, Cell Culture, Fluorescence, Transfection, Control, Plasmid Preparation, Staining