Journal: Acta Histochemica et Cytochemica

Article Title: Close Association of Aquaporin-2 Internalization with Caveolin-1

doi: 10.1267/ahc.12003

Figure Lengend Snippet: Colocalization of AQP2 and caveolin-1. Confluent MDCK-hAQP2 cells grown on permeable supports without any treatment as a control ( a–c ), treated with forskolin for 30 min ( d–f ), or treated with forskolin for 30 min, washed, and then incubated without forskolin for 30 min ( g–i ) were fixed. Cells were sectioned perpendicular to the supports with a cryostat and double-immunofluorescently labeled for AQP2 (red) and caveolin-1 (green). Nuclei were stained with DAPI (blue). Merged confocal images are shown on the left. In the control, AQP2 is seen in the subapical cytoplasm and seems not to colocalize with caveolin-1, which is present on the apical membrane. AQP2 is largely colocalized with caveolin-1 on the apical membrane upon forskolin treatment. Some AQP2 internalized after washout is colocalized with caveolin-1. Bar=10 µm.

Article Snippet: Primary antibodies were as follows: rabbit anti-AQP2 [ , ], guinea pig anti-AQP2 [ , ], goat anti-AQP2 (sc-9882; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Rab11a (Zymed, San Francisco, CA), mouse anti-EEA1 (clone 14; BD Biosciences, San Jose, CA), goat anti-EEA1 (sc-6414, Santa Cruz Biotechnology), mouse anti-caveolin-1 (clone 2234, BD Biosciences), rabbit anti-caveolin-1 (sc-894, Santa Cruz Biotechnology), mouse anti-phosphorylated caveolin-1 (clone 56, BD Biosciences), rabbit anti-cathepsin D [ ], and mouse anti-gp135 [ ].

Techniques: Incubation, Labeling, Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Close Association of Aquaporin-2 Internalization with Caveolin-1

doi: 10.1267/ahc.12003

Figure Lengend Snippet: AQP2 is recovered in Triton X-100-insoluble fraction. Samples of Triton X-100-soluble (S) and -insoluble (I) fractions from MDCK-hAQP2 cell lysates with (+) or without (–) forskolin treatment for 30 min were subjected to SDS-PAGE and immunoblotting using anti-AQP2 and anti-caveolin-1 (Cav1) antibodies.

Article Snippet: Primary antibodies were as follows: rabbit anti-AQP2 [ , ], guinea pig anti-AQP2 [ , ], goat anti-AQP2 (sc-9882; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Rab11a (Zymed, San Francisco, CA), mouse anti-EEA1 (clone 14; BD Biosciences, San Jose, CA), goat anti-EEA1 (sc-6414, Santa Cruz Biotechnology), mouse anti-caveolin-1 (clone 2234, BD Biosciences), rabbit anti-caveolin-1 (sc-894, Santa Cruz Biotechnology), mouse anti-phosphorylated caveolin-1 (clone 56, BD Biosciences), rabbit anti-cathepsin D [ ], and mouse anti-gp135 [ ].

Techniques: SDS Page, Western Blot

Journal: Acta Histochemica et Cytochemica

Article Title: Close Association of Aquaporin-2 Internalization with Caveolin-1

doi: 10.1267/ahc.12003

Figure Lengend Snippet: AQP2 co-immunoprecipitates with caveolin-1. a : In immunoprecipitates with anti-AQP2, caveolin-1 is detected as a dense band in forskolin-treated cells (F) and a faint band in control cells (C) at 22–24 kDa (arrow) with anti-caveolin-1 (Cav1). Dense bands both around 20 kDa (arrowhead) and 40 kDa (double-arrowhead) seem to be non-specific because these bands are also detected in the lane in which rabbit IgG was electrophoresed (RbIgG). b : Immunoprecipitates with anti-AQP2 from each cell lysate described as follows were subjected to immunoblotting with anti-caveolin-1 (Cav1). –: control cells, +: cells treated with forskolin for 30 min, ±: cells treated with forskolin for 30 min, washed, and then incubated without forskolin for 30 min.

Article Snippet: Primary antibodies were as follows: rabbit anti-AQP2 [ , ], guinea pig anti-AQP2 [ , ], goat anti-AQP2 (sc-9882; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Rab11a (Zymed, San Francisco, CA), mouse anti-EEA1 (clone 14; BD Biosciences, San Jose, CA), goat anti-EEA1 (sc-6414, Santa Cruz Biotechnology), mouse anti-caveolin-1 (clone 2234, BD Biosciences), rabbit anti-caveolin-1 (sc-894, Santa Cruz Biotechnology), mouse anti-phosphorylated caveolin-1 (clone 56, BD Biosciences), rabbit anti-cathepsin D [ ], and mouse anti-gp135 [ ].

Techniques: Western Blot, Incubation

Journal: Acta Histochemica et Cytochemica

Article Title: Close Association of Aquaporin-2 Internalization with Caveolin-1

doi: 10.1267/ahc.12003

Figure Lengend Snippet: Internalized caveolin-1 is phosphorylated. Samples of Triton X-100-insoluble fractions from MDCK-hAQP2 cell lysates were subjected to SDS-PAGE and immunoblotting using anti-phosphorylated caveolin-1 antibody. A strong band is detected in cells treated with forskolin and subsequently washed and incubated without forskolin for 30 min (±). Faint bands are detected in cells both treated (+) and untreated (–) with forskolin.

Article Snippet: Primary antibodies were as follows: rabbit anti-AQP2 [ , ], guinea pig anti-AQP2 [ , ], goat anti-AQP2 (sc-9882; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Rab11a (Zymed, San Francisco, CA), mouse anti-EEA1 (clone 14; BD Biosciences, San Jose, CA), goat anti-EEA1 (sc-6414, Santa Cruz Biotechnology), mouse anti-caveolin-1 (clone 2234, BD Biosciences), rabbit anti-caveolin-1 (sc-894, Santa Cruz Biotechnology), mouse anti-phosphorylated caveolin-1 (clone 56, BD Biosciences), rabbit anti-cathepsin D [ ], and mouse anti-gp135 [ ].

Techniques: SDS Page, Western Blot, Incubation

Journal: Acta Histochemica et Cytochemica

Article Title: Close Association of Aquaporin-2 Internalization with Caveolin-1

doi: 10.1267/ahc.12003

Figure Lengend Snippet: AQP2 is internalized to the same compartment with caveolin-1 and flotillin-2. ( a–f ) MDCK-hAQP2 cells were seeded on coverslips and subjected to forskolin treatment for 30 min ( a, d ) and subsequent washing and incubation without forskolin for 30 min ( b, e ) or for 2 hr ( c, f ). Double-immunofluorescence labeling for AQP2 and EEA1 ( a–c ) or for AQP2 and caveolin-1 ( d–f ) was carried out and their localization was observed with a laser confocal microscope. Projection images of 4 consecutive confocal images (0.4 µm intervals) are shown. AQP2 is shown in red. EEA1 and caveolin-1 are shown in green. An enlarged view of the rectangle area is shown in the inset ( f ). Both AQP2 and caveolin-1 are internalized in the EEA1-positive compartment 30 min after washing and then differentially localized at 2 hr. g, h : MDCK-hAQP2 cells transiently transfected with EGFP-flotillin-2 were treated with forskolin for 30 min ( g ) and then washed and incubated without forskolin for 30 min ( h ). Localization of AQP2 and flotillin-2 was observed with a laser confocal microscope. Projection images of 4 consecutive confocal images (0.4 µm intervals) are shown. AQP2 is shown in red. EGFP fluorescence is shown in green. Both AQP2 and EGFP-flotillin-2 are internalized in the same compartment. Bars=10 µm (5 µm in inset).

Article Snippet: Primary antibodies were as follows: rabbit anti-AQP2 [ , ], guinea pig anti-AQP2 [ , ], goat anti-AQP2 (sc-9882; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Rab11a (Zymed, San Francisco, CA), mouse anti-EEA1 (clone 14; BD Biosciences, San Jose, CA), goat anti-EEA1 (sc-6414, Santa Cruz Biotechnology), mouse anti-caveolin-1 (clone 2234, BD Biosciences), rabbit anti-caveolin-1 (sc-894, Santa Cruz Biotechnology), mouse anti-phosphorylated caveolin-1 (clone 56, BD Biosciences), rabbit anti-cathepsin D [ ], and mouse anti-gp135 [ ].

Techniques: Incubation, Immunofluorescence, Labeling, Microscopy, Transfection, Fluorescence

Journal: Acta Histochemica et Cytochemica

Article Title: Close Association of Aquaporin-2 Internalization with Caveolin-1

doi: 10.1267/ahc.12003

Figure Lengend Snippet: Ultrastructural localization of AQP2 and caveolin-1. MDCK-hAQP2 cells were treated with forskolin for 30 min ( a ), and then washed and incubated without forskolin for 30 min ( b ). Ultrathin cryosections were double-labeled for AQP2 (12 nm colloidal gold) and caveolin-1 (6 nm colloidal gold). In forskolin-treated cells ( a ), some AQP2 is colocalized with caveolin-1 at caveolar structures in the apical membrane domain (arrows). AQP2 on the microvillous membrane is not colocalized with caveolin-1. When cells were washed and incubated without forskolin for 30 min ( b ), AQP2 was colocalized with caveolin-1 in the intracellular vesicles (arrows). Bars=100 nm.

Article Snippet: Primary antibodies were as follows: rabbit anti-AQP2 [ , ], guinea pig anti-AQP2 [ , ], goat anti-AQP2 (sc-9882; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Rab11a (Zymed, San Francisco, CA), mouse anti-EEA1 (clone 14; BD Biosciences, San Jose, CA), goat anti-EEA1 (sc-6414, Santa Cruz Biotechnology), mouse anti-caveolin-1 (clone 2234, BD Biosciences), rabbit anti-caveolin-1 (sc-894, Santa Cruz Biotechnology), mouse anti-phosphorylated caveolin-1 (clone 56, BD Biosciences), rabbit anti-cathepsin D [ ], and mouse anti-gp135 [ ].

Techniques: Incubation, Labeling

Journal: Journal of Biomedical Materials Research. Part B, Applied Biomaterials

Article Title: Safety of intradiscal injection and biocompatibility of polyester amide microspheres in a canine model predisposed to intervertebral disc degeneration

doi: 10.1002/jbm.b.33579

Figure Lengend Snippet: A–G. Relative gene expression levels in non, sham‐, and PEAM‐injected, canine IVDs at 6 months follow‐up. The noninjected nuclei pulposi (NP) are set at 1. A–C. Gene expression levels of COL2A1 ( A ) and COL1A1 ( B ) were not significantly different between conditions, neither was collagen corrected for DNA ( C ). D. CASP3 expression levels were significantly lower in the AF compared with the NP. E. Expression levels of CAV1 were significantly higher in sham‐injected IVDs (NP + AF) compared with noninjected control IVDs. F. In all conditions glycosaminoglycan (GAG) corrected for DNA were significantly higher in the NP compared with the AF. G. The PEAM‐injected IVDs showed a significant higher BAX/BCL2 ratio compared with the sham‐injected IVDs. Data in A , B , D , and E are expressed as n ‐fold changes, in C and F as mean values, and in G as ratio ± standard deviation. * Indicate significant difference at a 99% confidence level

Article Snippet: Paraffin sections (5 µm) stained with hematoxylin/eosin and with picrosirius red/alcian blue were histopathologically evaluated by two independent investigators, blinded to the treatments, according to the grading scheme developed by Bergknut et al. Immunohistochemistry for caveolin‐1 [monoclonal mouse anti‐caveolin‐1 antibody (Clone 2297, 610406, BD Biosciences) diluted 1:50 in PBS] was performed as described previously.

Techniques: Expressing, Injection, Standard Deviation

Journal: Cell metabolism

Article Title: Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier

doi: 10.1016/j.cmet.2019.11.002

Figure Lengend Snippet: (A) Macroscopic tumor burden was assessed in Mpc1Lrig1KO, Mpc1Lrig1KO/+, and control mice at 100 days post-AOM (n=28–45 mice), ****p≤0.0001, ***p≤0.001, one-way ANOVA with multiple comparisons. Representative gross colon tumor burden and distribution are shown (ruler tick = 1mm). (B) At 100 days post-AOM, microscopic tumor grade and size assessed by pathologic grading in colons of Mpc1Lrig1KO, Mpc1Lrig1KO/+, and control mice (n=10–12), n.s.=not significant, *p≤0.05, one-way ANOVA with multiple comparisons on number of microscopic tumors per mouse. (C) Hematoxylin/Eosin and anti-β-catenin staining of control and Mpc1Lrig1KO adenomas at 100 days post-AOM, (100X, scale bars = 25μm). Black arrows indicate nuclear β-catenin. Green dashed circles indicate crypt necrosis. (D) Immunoblot of paired normal crypts (“C”) and tumor (“T”) samples from two control mice and two Mpc1Lrig1KO mice for MPC1, MPC2, and actin. Densitometry for MPC1 and MPC2, normalized to actin is shown with relative values adjusted to 100 for each control mouse normal crypt sample. (E) 100 days post-AOM, Ki67-positive nuclei as a percentage of total adenoma nuclei of Mpc1Lrig1KO and control adenomas were quantified on blinded high magnification images (n =5–7 mice, adenomas=10–20), **p≤0.01, unpaired two-tailed t-test. Representative images to the right (100X, scale bars=25μm). (F) 100 days post-AOM, cleaved caspase 3-positive nuclei of ApcLrig1KO/+Mpc1Lrig1KO and ApcLrig1KO/+ adenomas (black arrows) were quantified on blinded high magnification images (n =5–7 mice, adenomas=13–15, 100X, scale bars=25 μm), n.s.=not significant, unpaired two-tailed t-test. Green dashed circle indicates crypt necrosis and these positive nuclei were omitted. (G) At 49 days post-AOM, regions of superficial inflammation, epithelial injury with active inflammation (AI), aberrant foci, and low-grade dysplasia assessed by pathologic grading in colons of Mpc1Lrig1KO/+, Mpc1Lrig1KO/+, and control mice (n=8–10 mice), n.s.=not significant, unpaired t-test on number of microscopic lesions per mouse.

Article Snippet: b-catenin (IHC) 1:200 , BD Biosciences #610153 , RRID:AB_397554.

Techniques: Staining, Western Blot, Two Tailed Test

Journal: Cell metabolism

Article Title: Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier

doi: 10.1016/j.cmet.2019.11.002

Figure Lengend Snippet: (A) Macroscopic tumor burden was assessed in Apc Lrig1KO/+Mpc1Lrig1KO, Apc Lrig1KO/+Mpc1Lrig1KO/+, and Apc Lrig1KO/+ mice at 90 days post-TAM (n=11–12 mice), **p≤0.01, *p≤0.05, one-way ANOVA with multiple comparisons (B) Microscopic tumor grade and size assessed by pathologic grading in the small intestine (left) and the colon (right) for ApcLrig1KO/+ Mpc1Lrig1KO and ApcLrig1KO/+ mice (n=5–7), n.s.=not significant, *p≤0.05, unpaired t-test on number of microscopic tumors per mouse. (C) Hematoxylin/Eosin, and anti-β-catenin staining of ApcLrig1KO/+ and ApcLrig1KO/+ Mpc1Lrig1KO adenomas (100X, scale bars=25 μm). Black arrows indicate nuclear β-catenin. (D) Immunoblot of paired normal crypts (“C”) and tumor (“T”) samples from two ApcLrig1KO/+ mice and two ApcLrig1KO/+Mpc1Lrig1KO mice for MPC1, MPC2, and actin. Densitometry for MPC1 and MPC2, normalized to actin is shown with relative values adjusted to 100 for each ApcLrig1KO/+ mouse normal crypt sample. (E) 90 days post-tamoxifen, Ki67-positive nuclei as a percentage of total adenoma nuclei of ApcLrig1KO/+Mpc1Lrig1KO and ApcLrig1KO/+ adenomas were quantified on blinded high magnification images (n =3–5 mice, adenomas=10–23), n.s.=not significant, unpaired two-tailed t-test. Representative images to the right (100X, scale bars=25 μm). (F) 90 days post-tamoxifen, cleaved caspase 3-positive nuclei of ApcLrig1KO/+Mpc1Lrig1KO and ApcLrig1KO/+ adenomas (black arrows) were quantified on blinded high magnification images (n =3–5 mice, adenomas = 15–16), n.s.= not significant, unpaired two-tailed t-test. Representative images to the right (100X, scale bars = 25 μm). Green dashed circle indicates crypt necrosis and these positive nuclei were omitted. (G) At 140 days post-TAM, macroscopic tumor burden per mouse in ApcVillKO/+ and ApcVillKO/+Mpc1VillKO was assessed (n=18–27,28: one small intestine was damaged during dissection and removed from analysis). *p≤0.05, unpaired t-test. (H) Microscopic tumor grade and size assessed by pathologic grading in the small intestine (left) and colon (right) of ApcVillKO/+ and ApcVillKO/+Mpc1VillKO mice (n=7–8), *p≤0.05, unpaired t-test on number of microscopic tumors per mouse.

Article Snippet: b-catenin (IHC) 1:200 , BD Biosciences #610153 , RRID:AB_397554.

Techniques: Staining, Western Blot, Two Tailed Test, Dissection

Journal: Cell metabolism

Article Title: Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier

doi: 10.1016/j.cmet.2019.11.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: b-catenin (IHC) 1:200 , BD Biosciences #610153 , RRID:AB_397554.

Techniques: Western Blot, Recombinant, Plasmid Preparation, Protease Inhibitor, Generated, Software