Journal: Nutrients

Article Title: Vitamin C: A Novel Regulator of Neutrophil Extracellular Trap Formation

doi: 10.3390/nu5083131

Figure Lengend Snippet: Autophagy signaling is induced in Vitamin C deficient neutrophils. ( A ) Real time QPCR for ATG3, ATG5, ATG6, ATG7, and ATG8 mRNA from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice, ( N = 6 for each group, * p < 0.05). ( B ) Representative Western blot for expression of LC3B-I and LC3B-II from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice. Densitometry of LC3B-II/actin from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice ( N = 6 for each group, * p < 0.05). ( C ) Representative Western blot for expression of p62 and actin from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice. Densitometry of normalized p62 expression from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice ( N = 6 for each group, ns p = 0.3).

Article Snippet: Purified rabbit polyclonal antibodies to LC3B (L7543, Sigma-Aldrich), cleaved caspase-3 (#9661, Cell Signaling), caspase-3 (#9662, Cell Signaling), p62/SQSTM1 (NBP1-48320, Novus Biologicals), NFκB p65 (sc-109, Santa Cruz Biotechnology), Lamin B (sc-6216, Santa Cruz Biotechnology), and actin (sc-1616, Santa Cruz Biotechnology) were used in this study.

Techniques: Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: Primers

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: BGJ398 reduces tumor burden in an oncogene-driven murine model of CCA. A, FGFRs are up-regulated in a YAP-driven murine model of CCA. mRNA expression of Fgfr1, Fgfr2, Fgfr3, and Fgfr4 using qPCR and RNA sequencing of mouse tumors compared with adjacent liver. Thr dashed line represents adjacent liver, which served as the control. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, representative immunostaining images for phospho-FRS2 in vehicle (Veh)- and BGJ398-treated animals. Scale bars: 50 μm. C, liver appearance of mice after intrabiliary injection of myr-Akt and YapS127A Sleeping Beauty transposon-transposase complexes coupled with lobar bile duct ligation and daily intraperitoneal injections of IL-33 (1 μg for 3 days) with (right panel) and without (left panel) BGJ398 treatment (12.5 mg/kg/day) for 2 weeks. D, ratio of tumor weight to liver weight of the ligated lobe expressed as a percentage in vehicle (n = 9)- and BGJ398 (n = 6)-treated animals. *, p < 0.05. E, number of nodules in vehicle (n = 8)- and BGJ398 (n = 6)-treated animals with tumors. *, p < 0.05. F, representative photomicrographs of hematoxylin and eosin-stained tumor sections and adjacent liver are shown in vehicle- and BGJ398-treated animals. Scale bars: 100 μm. G, apoptotic cells were quantified by counting TUNEL-positive nuclei in five random microscopic fields (×20) using a fluorescent microscope. Shown are images (top panel) and the percentage of TUNEL-positive cells (bottom panel) in representative sections of vehicle- and BGJ398-treated animals. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. H, immunofluorescence images (top panel) and percentage of Ki67-positive cells (bottom panel) in representative sections of vehicle- and BGJ398-treated animals. Mean ± S.E. are depicted for n = 3. Representative immunofluorescence experiments included tissue sections from three mice from each group. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, RNA Sequencing Assay, Immunostaining, Injection, Ligation, Staining, TUNEL Assay, Microscopy, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma *

doi: 10.1074/jbc.M115.698472

Figure Lengend Snippet: BGJ398 inhibits YAP activation in a PDX model of CCA. A, representative immunostaining images for nuclear YAP (brown staining) in YAP-positive (PDX1) and YAP-negative (PDX2) tumors. B, mRNA expression of Yap, Ctgf, and Sox4 in PDX1 and PDX2. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. C, mRNA expression of Fgfr1, Fgfr2, Fgfr3, and Fgfr4 in PDX1 and PDX2. Mean ± S.E. are depicted for n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, tumor weight in mg of PDX1 (left panel) and PDX2 (right panel) mice treated for 2 weeks with vehicle (n = 5) or 12.5 mg/kg/day BGJ398 (n = 5). *, p < 0.05. E, representative photomicrographs of hematoxylin and eosin-stained tumors in vehicle- and BGJ398-treated PDX1 (left panel) and PDX2 (right panel) animals. Scale bars: 1 mm. F, immunofluorescence images of CK-19 staining (to outline the biliary epithelium) and YAP in tissue sections obtained from PDX1 mice treated with vehicle or 12.5 mg/kg BGJ398 for 2 weeks. G, mRNA expression of Yap, Ctgf, Sox4, and Mcl-1 in PDX1 animals treated with vehicle or 12.5 mg/kg/day BGJ398 for 2 weeks. Mean ± S.E. are depicted for n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. H, fluorescence images (left panel) and percentage of TUNEL-positive cells (right panel) in representative sections of vehicle- and BGJ398-treated PDX1 (top panel) and PDX2 (bottom panel) animals. Apoptotic cells were quantified by counting TUNEL-positive nuclei in five random microscopic fields (×20) using a fluorescent microscope. Mean ± S.E. are depicted for n = 3. ***, p < 0.001. I, immunofluorescence images (left panel) and percentage of Ki67-positive cells (right panel) in representative sections of vehicle- and BGJ398-treated PDX1 (top panel) and PDX2 (bottom panel) animals. Mean ± S.E. are depicted for n = 3. Representative immunofluorescence experiments included tissue sections from three mice from each group. Scale bars: 50 μm.

Article Snippet: The following primary antibodies were used for immunoblot analysis: phospho-YAPY357 (ab62751) from Abcam; α-tubulin (CST 2144), FGFR1 (CST 9740P), FGFR2 (CST 11835S), FGFR4 (CST 8562P), GAPDH (Millipore MAB374), histone H3 (CST 9715), LATS1 (CST 66B5), LATS2 (CST 13646), Mcl-1 (CST 4572), MST1 (CST 3682), MST2 (CST 3952), phospho-YAPS127 (CST 4911S), TAZ (CST 4883), and YAP (CST 4912) from Cell Signaling Technology; and β-actin (SC-1615), FGFR3 (SC-13121), and T-box 5 (TBX5; SC-17866) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Immunostaining, Staining, Expressing, Immunofluorescence, Fluorescence, TUNEL Assay, Microscopy

Journal: Molecular neurobiology

Article Title: Citalopram reduces aggregation of ATXN3 in a YAC transgenic mouse model of Machado-Joseph disease

doi: 10.1007/s12035-018-1331-2

Figure Lengend Snippet: Assessment of macroautophagy markers shows increased levels of p62 in the spinal cord (B), and of p62 and beclin-1 in the cerebellum (C) of citalopram-treated mice compared to controls. Citalopram treatment did not affect levels of LC3 in any of the three regions (A-C). (A, B, and C) Left panels show immunoblots of indicated proteins in soluble fractions of brainstem, spinal cord and cerebellum. Right panels display quantification of band intensity, with values normalized to Gapdh. Bars represent the average percentage of protein relative to vehicle-treated mice (± SEM). Statistical significance is indicated as *P < 0.05 and **P < 0.01. Mice 1, 2, and 20 were excluded from statistical analysis. Diamond (◆) represents p62 bands.

Article Snippet: Total protein lysates from soluble fractions (100 μg from brainstem, and cerebellum) were resolved on 10% SDS-PAGE gels, and corresponding PVDF membranes were incubated overnight at 4 °C with primary antibodies: mouse anti-HSP70 (1:500; SPA810, Enzo Life Sciences), rabbit anti-HSP90α (1:1000; ab2928, Abcam), mouse anti-HSP90β (1:1000; ADI-SPA842, Enzo Life Sciences), rabbit anti-HSP40 (1:1000; #4868, Cell Signaling), mouse anti-p62/SQSTM1 (1:1000; H00008878-M01, Novus Biologicals), rabbit anti-Beclin1 (1:1000; ab207612, Abcam), rabbit anti-LC3 (1:500; PM036, MBL International Corporation), mouse anti-GAPDH (1:5000; MAB374, Millipore), and rabbit anti-MJD [ 30 ] (1:30000).

Techniques: Western Blot