Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Npc1 deficiency impairs microglia function via TREM2-mTOR signaling in Niemann-Pick disease type C.

doi: 10.1016/j.bbadis.2024.167478

Figure Lengend Snippet: Fig. 1. Loss of Npc1 impairs brain development in mouse. A Representative photograph of brain from Npc1−/−mice and littermate control at postnatal day 63 (P63). B Brain weight curves of Npc1−/−mice and littermate controls (n = 9; Page x genotype < 0.0001, Page < 0.0001, Pgenotype < 0.0001). C Representative coronal sections of Npc1−/−mice and littermate controls at P42 visualized by hematoxylin and eosin staining. D Quantification of the cortical thickness of mice from P1 to P63 (n = 3). E, F Representative confocal images and quantification of Filipin (white) and DAPI (blue) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). G, H Representative confocal images and quantification of GFAP (red) and DAPI (blue) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). I, J Repre sentative confocal images and quantification of MBP (green) and DAPI (blue) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). K, L Representative confocal images and quantification of cleaved-Caspase3 (c.CASP3, green) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). M Western blot analysis for c.CASP3, BAX and BCL2 protein expression in the brain of Npc1−/−mice and littermate controls (n = 3). Results are presented as Mean ± SEM. n.s., not sig nificant. *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary and secondary antibodies were used as follows: cleaved-Caspase3 (Cell Signaling Technology, USA; Cat No. 9664), BAX (Cell Signaling Technology, USA; Cat No. 14796), BCL2 (BOSTER, China; Cat No. BA0412), APOE (Abcam, UK; Cat No. ab183597), CTSD (Abcam, UK; Cat No. ab75852), Npc1 (Abcam, UK; Cat No. ab134113), Npc2 (Abcam, UK; Cat No. ab218192), TREM2 (Cell Signaling Technology, USA; Cat No. 76765), TREM2 (R&D Systems, USA; Cat No. AF1729), TREM2 (Abcam, UK; Cat No. ab305103), SYK (Cell Signaling Technology, USA; Cat No.13198), p-SYK (Cell Signaling Technology, USA; Cat No.2710), AKT (Cell Signaling Technology, USA; Cat No.4691), p-AKT (Cell Signaling Technology, USA; Cat No.13038), GSK3β (Cell Signaling Technology, USA; Cat No. 12456), p-GSK3β (Cell Signaling Technology, USA; Cat No. 5558), mTOR (Cell Signaling Technology, USA; Cat No. 2983), p-mTOR (Cell Signaling Technology, USA; Cat No. 5536), S6K1 (Cell Signaling Technology, USA; Cat No. 34475), p-S6K1 (Cell Signaling Technology, USA; Cat No. 9234), 4EBP1 (Cell Signaling Technology, USA; Cat No. 9644), p-4EBP1 (Cell Signaling Technology, USA; Cat No. 9451), CD63 (Abcam, UK; Cat No. ab217345), GFAP (Cell Signaling Technology, USA; Cat No. 80788), β-Actin (Affinity Biosciences, USA; Cat No. AF7018), β-Tubulin (Absin, China; Cat No. abs830032), Goat anti-Rabbit IgG-HRP (Absin, China; Cat No. abs20040), Goat anti-Mouse IgG-HRP (Absin, China; Cat No. abs20039), Rabbit anti-Sheep IgG-HRP (Abcam, UK; Cat No. ab6747).

Techniques: Control, Staining, Western Blot, Expressing

Journal: Genetics and molecular research : GMR

Article Title: Increased survivin expression and its association with clinical parameters of congenital choledochal cysts.

doi: 10.4238/gmr.15028032

Figure Lengend Snippet: Figure 1. Immunohistochemical staining results for the protein expression of survivin. The cytoplasm showed high survivin expression, which presented as granular form, with yellow or dark brown staining, while the nuclei were hardly stained (A. cytoplasm, 200X; B. nuclei, 400X).

Article Snippet: The primary mouse anti-human survivin monoclonal antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (China).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: Inhibition of BCL-XL but not inhibition of BCL-2 cooperates with PLX4032 in the killing of BRAFV600E mutant melanoma cell lines. (a) M14, UACC257, Malme3M and SKMEL5 BRAFV600E cell lines were treated with DMSO (vehicle; -) or PLX4032 (+ at 3 μM) in the presence of the pan-caspase inhibitor Q-VD-OPh for 24 h before western blot analysis to detect the indicated pro-survival BCL-2-like proteins. Probing with an antibody to HSP70 was used as a loading control. (b) BRAFV600E mutant melanoma cell lines from panel (a) were treated with DMSO, PLX4032 (3 μM) and/or (c) ABT-737 (1 μM; inhibitor of BCL-2, BCL-XL and BCL-W), ABT-199 (1 μM; inhibitor of BCL-2) or ABT-1155463 (1 μM; inhibitor of BCL-XL). Cell survival was assessed after 5 days of treatment by flow cytometric analysis. Living cells were identified as those negative for Annexin V and propidium iodide. The data are derived from three independent experimental replicates, each comprising the average of three technical replicates and are presented as mean±S.E.M.; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Inhibition, Mutagenesis, Western Blot, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: HGF inhibits PLX4032-induced upregulation of PUMA and BIM in cMET+ BRAF mutant melanoma cells and thereby protects them against PLX4032-induced killing. (a) cMET− (M14 and UACC257) and cMET+ (Malme3M and SKMEL5) BRAFV600E melanoma cell lines were treated with DMSO (vehicle; black bar) or PLX4032 (grey bars, at 3 μM) with the indicated concentrations of HGF (0, 3, 6, 12, 25, 50, 100, 200 ng/ml) for 5 days and cell survival was assessed by flow cytometry. Living cells were defined as those negative for Annexin V and propidium iodide. The concentration at which HGF causes significant protection from PLX4032-induced killing is indicated. Data are derived from three independent experimental replicates, each comprising the average of three technical replicates and are presented as mean±S.E.M.; *P<0.05, **P<0.01, ****P<0.0001. (b) cMET− (M14 and UACC257) and cMET+ (Malme3M and SKMEL5) BRAF mutant cells were treated with single agents or combinations of DMSO (vehicle), HGF (50 ng/ml) and PLX4032 (3 μM) for 24 h in the presence of Q-VD-OPh. The mRNA of cells was subjected to qRT-PCR analysis to measure the expression of pro-survival (BCL-2, BCL-X, BFL-1, MCL-1) and pro-apoptotic (BIM, PUMA) BCL-2 family members. The mRNA expression levels of the different genes were normalized to the levels of Gapdh and the data are presented as fold change relative to DMSO (vehicle)-treated control cells. The data are derived from three independent experimental replicates and are presented as mean±S.E.M. (c) Cells were treated as described in panel (b) and analyzed by western blotting for the expression of pro-survival (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, PUMA) BCL-2 family members. Probing for HSP70 was used as a loading control

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Mutagenesis, Flow Cytometry, Concentration Assay, Derivative Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: The combination of PLX4032 and BCL-XL inhibition can partially overcome HGF-mediated resistance of cMET+ BRAF mutant melanoma cells. (a) Malme3M and SKMEL5 cells were treated with DMSO (vehicle), PLX4032 (3 μM) alone or in combination with ABT-737 (1 μM) (blue bars), ABT-199 (1 μM) (green bars), A-1155463 (1 μM) (red bars), with or without addition of HGF (50 ng/ml) for 5 days and cell survival was assessed by flow cytometry. Living cells were identified as those negative for Annexin V and PI. The data are derived from three independent experimental replicates, each comprising the average of three technical replicates. The data were analyzed using Student's t-test and are presented as mean±S.E.M.; *P<0.05, **P<0.01. (b) Under steady-state conditions, low levels of pro-apoptotic BH3-only proteins are present in the BRAF mutant melanoma cells. Hence, pro-survival proteins are able to neutralize BAX and BAK to cause survival of the cell. In the presence of PLX4032, PUMA and BIM are getting induced, which leads to sequestration of pro-survival proteins, allowing BAX and BAK to induce apoptosis. In the presence of HGF, PLX4032 mediated upregulation of PUMA and BIM is substantially diminished, and this is the underlying molecular mechanism that causes melanoma cells to develop resistance to PLX4032-induced killing. When combining PLX4032 and the BCL-XL-specific inhibitor, A-1155463, HGF-mediated resistance can partially be overcome

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Inhibition, Mutagenesis, Flow Cytometry, Derivative Assay