Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Diet-induced aortic valve disease in mice haploinsufficient for the Notch pathway effector RBPJK/CSL.

doi: 10.1161/ATVBAHA.111.227561

Figure Lengend Snippet: Figure 2. Greater leaflet thickness and increased macrophage deposition in RBPKO/ mice fed an HCVD diet. Shown are Masson Trichrome staining (A to F) of aortic valves indicating collagen deposition (arrows) and Mac3 immuno- staining (G to L) of aortic valves demon- strating macrophage infiltration (red; arrows indicate positive immunostaining) in WT mice fed the CC diet (A and G) or the HCVD diet (D and J), Notch1KO/ mice fed the CC diet (B and H) or the HCVD diet (E and K), and RBPKO/ mice fed the CC diet (C and I) or the HCVD diet (F and L). M and N, Mean data of cusp area (M) and percentage of positively stained sections for Mac3 (N) for all groups (n4 per group). One-way ANOVA and LSD post hoc analysis were used to compare differences between groups. Bars with different letters indi- cate significant differences (P0.05) vs all other groups (LSD post hoc analysis). Scale bar0.1 mm.

Article Snippet: Inflammation was detected with anti-Mac3 rat monoclonal antibody (SC19991, Santa Cruz Technol, 1:200).

Techniques: Staining, Immunostaining

Journal: OncoTargets and therapy

Article Title: PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2

doi: 10.2147/OTT.S237343

Figure Lengend Snippet: The expression of PHLPP in bortezomib-resistant MM cells. ( A ) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in bone marrow samples from patients with MM (upper), and quantification of the bands (lower). ( B ) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266 cells and bortezomib-resistant U266 cells (upper), and quantification of the bands (lower). MM-S, BTZ-sensitive MM; MM-R, BTZ-resistant MM; U266-R, BTZ-resistant U266 cells. * P < 0.05.

Article Snippet: The membranes were incubated with primary antibody: anti-LAMP2 antibody (1:1000, cat no. 49067, Cell Signaling Technology), anti-LAMP1 antibody (1:1000, cat no. ab13523, Abcam), anti-LAMP2A antibody (1:1000, cat no. ab125068, Abcam), anti-LAMP2B antibody (1:1000, cat no. ab18529, Abcam), anti-AKT antibody (1:1000, cat no. ab18785, Abcam), anti-phospho-Akt (Ser473) antibody (1:1000, cat no. 4058, Cell Signaling Technology), anti-PHLPP antibody (1:1000, cat no. ab62830, Abcam); anti-Beclin1 antibody (1:1000, cat no. ab210498, Abcam); anti-Atg5 antibody (1:1000, cat no. ab228668, Abcam); anti-p62 antibody (1:1000, cat no. ab56416, Abcam); anti-LC3B antibody (1:1000, cat no. ab51520, Abcam).

Techniques: Expressing, Western Blot

Journal: OncoTargets and therapy

Article Title: PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2

doi: 10.2147/OTT.S237343

Figure Lengend Snippet: Overexpression of PHLPP sensitizes MM cells to BTZ. ( A ) Western blot analyses of PHLPP expression in U266 cells and BTZ-resistant U266 cells after lentivirus infection. ( B ) BrdU assays were used to determine cell viability after sh-PHLPP or PHLPP lentivirus infection in U266 and U266-R cells, respectively. ( C ) Flow cytometry was used to determine apoptosis after knockdown or overexpression of PHLPP under BTZ treatment. ( D ) U266 cells were infected with PHLPP lentivirus and were then injected into nude mice. Tumor volumes were measured weekly. ( E ) PHLPP and LAMP2 expression in tumor sections were evaluated using immunohistochemistry (IHC); Magnification, 100X; * P < 0.05.

Article Snippet: The membranes were incubated with primary antibody: anti-LAMP2 antibody (1:1000, cat no. 49067, Cell Signaling Technology), anti-LAMP1 antibody (1:1000, cat no. ab13523, Abcam), anti-LAMP2A antibody (1:1000, cat no. ab125068, Abcam), anti-LAMP2B antibody (1:1000, cat no. ab18529, Abcam), anti-AKT antibody (1:1000, cat no. ab18785, Abcam), anti-phospho-Akt (Ser473) antibody (1:1000, cat no. 4058, Cell Signaling Technology), anti-PHLPP antibody (1:1000, cat no. ab62830, Abcam); anti-Beclin1 antibody (1:1000, cat no. ab210498, Abcam); anti-Atg5 antibody (1:1000, cat no. ab228668, Abcam); anti-p62 antibody (1:1000, cat no. ab56416, Abcam); anti-LC3B antibody (1:1000, cat no. ab51520, Abcam).

Techniques: Over Expression, Western Blot, Expressing, Infection, Flow Cytometry, Knockdown, Injection, Immunohistochemistry

Journal: OncoTargets and therapy

Article Title: PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2

doi: 10.2147/OTT.S237343

Figure Lengend Snippet: PHLPP positively regulates LAMP2 expression. ( A ) Immunofluorescence assays were performed to investigate the interactions between PHLPP and LAMP2 in U266 cells. ( B ) Immunoprecipitation confirmed the interactions between PHLPP and LAMP2 in U266 cells; ( C ) EGFP-PHLPP was expressed in U266 cells for 48 hrs and loaded with lysotracker-Red DND-99 for 30 mins at 37°C. Cells were fixed and analyzed by confocal microscopy. ( D ) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266 and U266-R cells after infection with sh-PHLPP or PHLPP lentivirus. ( E ) Quantification of the bands in ( D ). * P < 0.05.

Article Snippet: The membranes were incubated with primary antibody: anti-LAMP2 antibody (1:1000, cat no. 49067, Cell Signaling Technology), anti-LAMP1 antibody (1:1000, cat no. ab13523, Abcam), anti-LAMP2A antibody (1:1000, cat no. ab125068, Abcam), anti-LAMP2B antibody (1:1000, cat no. ab18529, Abcam), anti-AKT antibody (1:1000, cat no. ab18785, Abcam), anti-phospho-Akt (Ser473) antibody (1:1000, cat no. 4058, Cell Signaling Technology), anti-PHLPP antibody (1:1000, cat no. ab62830, Abcam); anti-Beclin1 antibody (1:1000, cat no. ab210498, Abcam); anti-Atg5 antibody (1:1000, cat no. ab228668, Abcam); anti-p62 antibody (1:1000, cat no. ab56416, Abcam); anti-LC3B antibody (1:1000, cat no. ab51520, Abcam).

Techniques: Expressing, Immunofluorescence, Immunoprecipitation, Confocal Microscopy, Western Blot, Infection

Journal: OncoTargets and therapy

Article Title: PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2

doi: 10.2147/OTT.S237343

Figure Lengend Snippet: LAMP2 knockdown reverses the inhibitory effects of PHLPP in U266-R cells. ( A ) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266-R cells infected with sh-LAMP2 or PHLPP lentivirus under 40 nM BTZ treatment (left), and quantification of the bands (right). ( B ) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266 cells that were infected with LAMP2 or shPHLPP lentivirus under 5 nM BTZ treatment (left), and quantification of the bands (right). ( C ) Cell proliferation was evaluated using BrdU assay in U266-R cells that were infected with sh-LAMP2 or PHLPP lentivirus under 40 nM BTZ treatment. ( D ) Apoptosis was evaluated using flow cytometry in U266-R cells that were infected with sh-LAMP2 or PHLPP lentivirus under 40 nM BTZ treatment. ( E ) Cell proliferation was evaluated using BrdU assay in U266 cells that were infected with LAMP2 or shPHLPP lentivirus under 5 nM BTZ treatment. ( F ) Apoptosis was evaluated using flow cytometry in U266 cells that were infected with LAMP2 or shPHLPP lentivirus under 5 nM BTZ treatment. * P < 0.05.

Article Snippet: The membranes were incubated with primary antibody: anti-LAMP2 antibody (1:1000, cat no. 49067, Cell Signaling Technology), anti-LAMP1 antibody (1:1000, cat no. ab13523, Abcam), anti-LAMP2A antibody (1:1000, cat no. ab125068, Abcam), anti-LAMP2B antibody (1:1000, cat no. ab18529, Abcam), anti-AKT antibody (1:1000, cat no. ab18785, Abcam), anti-phospho-Akt (Ser473) antibody (1:1000, cat no. 4058, Cell Signaling Technology), anti-PHLPP antibody (1:1000, cat no. ab62830, Abcam); anti-Beclin1 antibody (1:1000, cat no. ab210498, Abcam); anti-Atg5 antibody (1:1000, cat no. ab228668, Abcam); anti-p62 antibody (1:1000, cat no. ab56416, Abcam); anti-LC3B antibody (1:1000, cat no. ab51520, Abcam).

Techniques: Knockdown, Western Blot, Expressing, Infection, BrdU Staining, Flow Cytometry