Journal: Biology Open

Article Title: Apolipoprotein CIII regulates lipoprotein-associated phospholipase A 2 expression via the MAPK and NFκB pathways

doi: 10.1242/bio.201410900

Figure Lengend Snippet: (A,B) THP-1 cells were incubated with the indicated concentrations of apo CIII for 48 h. Lp-PLA 2 expression was tested by western blotting ( n = 3). (C,D) THP-1 cells were incubated with 30 µg apo CIII for the indicated durations. The proteins were then collected, and Lp-PLA 2 expression was examined by western blotting. (E) THP-1 cells were incubated with 30 µg apo CIII for the indicated durations. Lp-PLA 2 mRNA was subsequently extracted and detected using quantitative PCR ( n = 3). (F,G) THP-1 cells were transfected with a human apo CIII vector for 48 h ( n = 3). The Lp-PLA 2 activity was assayed using the PAF-AH assay kit, and the Lp-PLA 2 mRNA level was determined using quantitative PCR. * P <0.05, ** P <0.01, *** P <0.0001.

Article Snippet: The plasma was analyzed using a PAF Acetylhydrolase kit (Cayman, USA) according to the manufacturer's instructions.

Techniques: Incubation, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Activity Assay

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a , b Stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify novel binding partners. MET wt-tGFP and MET N375S-tGFP cells were labeled with heavy (H) and light (L) amino acids. The cutoff values for SILAC ratios, after normalizing with MET expression, were set at (>2, <0.5) respectively. a Scatter plot of transformed MET wt /MET N375S ratios of membrane-bound proteins. Both axes represent MET wt (H)/MET N375S (L) and MET wt (L)/MET N375S (H) ratios, respectively. b List of various membranous proteins identified in SILAC analysis found to be associated with MET N375S . c Interaction of ectopic MET and endogenous HER2 in H2170 MET wt-tGFP and MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting. Left, input controls. Total MET and HER2 band intensities, normalized to input controls and relative to MET wt , are shown below ( n = 5). d , e Detection of MET/HER2 co-localization (red) in EV, MET wt-tGFP , and MET N375S-tGFP cells with proximity ligation assay (PLA). Representative images are shown ( d ), with the PLA signals quantified ( e ) and expressed as the number of signals/cell ± SD ( n > 3). Scale bar, 20 µm. f Representative confocal microscopy images of MET (Alexa-488; green) and HER2 (Alexa-594; red) in isogenic H2170 clones ( n = 2). DAPI (blue) was used as nuclear counter stain. Co-localized proteins appeared in yellow. The smaller panels are detailed views of the outlined (white) squares in the respective images. Scale bar, 20 µm. The total MET fluorescence that co-localized with HER2 signal in each variant was tabulated on the right, data presented as mean ± SD (five fields in one representative experiment). g MET/HER2 interaction in H2170 MET wt-tGFP and MET N375S-tGFP tumors shown in Fig. was detected with immunoprecipitation and immunoblotting. IgG was used as a loading control. h – k The role of Sema domain in MET N375S-tGFP cells was examined with recombinant Sema proteins, wild-type, rSema wt ; N375S mutant, rSema N375S . Cell viability of MET wt-tGFP ( h ) and MET N375S-tGFP ( i ) cells after treatment with 1, 5, 10 µg/ml of rSema wt or rSema N375S for 72 h, presented as mean ± SD ( n = 3). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. j Immunoblots showing the total and phosphorylated expressions of MET, HER2, Src, Akt, and ERK1/2 in lysates of the indicated cell lines after treatment with 10 µg/ml rSema. β-Actin was used as a loading control. k MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting after treatment with 10 µg/ml rSema. Left, input controls. l MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected after serum starvation (0.1% FBS), or co-incubated with HGF (0.1% FBS + 10 ng/ml HGF). Left, input controls. m HEK293 cells were transfected with 1 µg of either pCMV6-EV-tGFP vector, MET-wt, N375S, ∆Sema, N375Q, M1268T, or Y1248H plasmid, together with pCMV6-ERBB2-DDK plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Total MET and HER2 band intensities, relative to MET wt , are shown below ( n = 3). Left, input controls and phosphorylated proteins. β-Actin was used as a loading control.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Quantitative Proteomics, Cell Culture, Multiplex sample analysis, Binding Assay, Labeling, Expressing, Transformation Assay, Membrane, Immunoprecipitation, Western Blot, Proximity Ligation Assay, Confocal Microscopy, Clone Assay, Staining, Fluorescence, Variant Assay, Control, Recombinant, Mutagenesis, Two Tailed Test, Incubation, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a – c Isogenic H2170 MET N375S-tGFP cells were treated with vehicle (0.1% DMSO), MET inhibitor crizotinib (1 µM), HER2 inhibitor lapatinib (0.3 µM), or crizotinib/lapatinib combination. a Interaction of ectopic MET and endogenous HER2 was detected with immunoprecipitation and immunoblotting. Total MET and HER2 band intensities, relative to vehicle treated group, are shown below. Values represent average of three independent experiments. Left, input controls. b Detection of MET/HER2 co-localization (red) in MET N375S-tGFP cells with proximity ligation assay (PLA). Representative images are shown. Scale bar, 20 µm. c The PLA signals were quantified and expressed as number of signals/cell ± SD ( n > 3). Crizo, crizotinib; Lapa, lapatinib. d MET/HER2 interaction in H2170 MET N375S-tGFP tumors from Supplementary Fig. was detected with immunoprecipitation and immunoblotting after crizotinib treatment. Left, input controls. Cells were harvested 48 h after treatment, β-Actin was used as a loading control. e – h Immunohistochemistry staining of H2170 MET wt-tGFP and MET N375S-tGFP tumors from Supplementary Fig. . Representative images for p-MET ( e ) and p-HER2 ( g ) staining are shown. Expression of p-MET ( f ) and p-HER2 ( h ) were quantified and expressed as mean of positive-staining/100 cells ± SD ( n = 5). Scale bar, 50 µm. Two-tailed Student’s t test; * P < 0.05.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Control, Immunohistochemistry, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a – c Cellular invasion on isogenic wild-type (MET wt-tGFP ) and N375S mutant (MET N375S-tGFP ) clones, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. a H2170 MET N375S-tGFP cells were treated with crizotinib (10 µM), lapatinib (0.3 µM) or a crizotinib/lapatinib combination for 36 h. Representative images of cell invasion are shown. b H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 36 h. Representative images are shown. Scale bar: 200 μm. c Percentage of invaded cells relative to vehicle control (0.1% DMSO) or Scr siRNA control were expressed as mean ± SD ( n = 3). One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. Left, immunoblots of siRNA-treated H2170 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. d – f Calu-1 MET wt-tGFP and MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 24 h. Representative images of cell invasion for MET wt-tGFP ( d ) and MET N375S-tGFP cells ( e ) are shown. Scale bar: 200 μm. f Percentage of invaded cells relative to Scr siRNA control in Calu-1 MET wt-tGFP and MET N375S-tGFP cells were expressed as mean ± SD ( n = 3). Left, immunoblots of siRNA-treated Calu-1 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. g , h Anchorage-independent colony formation on isogenic H2170 MET N375S-tGFP cells, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. g H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in soft agar for 4 weeks. Representative images are shown. h The number of colonies were quantified after treatment with crizotinib, lapatinib, trastuzumab, or in combination. Data were presented as percentage of colonies relative to vehicle control (0.1% DMSO) ± SD ( n = 3). i – l Efficacies of HER2 inhibitors were evaluated in xenograft models. Tumor growth of MET wt-tGFP ( i ) and MET N375S-tGFP ( j ) xenografts after treatment with trastuzumab, pertuzumab, lapatinib, and ASLAN001 were expressed at mean ± SEM ( n = 5). Two-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. k Immunohistochemistry staining showing the changes in p-MET after treatment with trastuzumab in MET wt-tGFP and MET N375S-tGFP tumors. Representative images are shown. Scale bar, 50 µm. l Expression of p-MET was quantified and expressed at mean of positive-staining/100 cells ± SD ( n = 5). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. m HEK293 cells were transfected with 1 µg of either pCMV6-ERBB2-wt, ∆D1, ∆D2, ∆D3, ∆D4, or ∆TK plasmid, together with pCMV6-MET-N375S plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Left, input controls and phosphorylated proteins. β-Actin was used as a loading control. n , o Isogenic Calu-1 MET wt-tGFP ( n ) and MET N375S-tGFP ( o ) cells were engrafted into SCID mice, and treated daily with vehicle ( n = 6) or 15 mg/kg afatinib ( n = 5) starting 14 days after inoculation. Arrows indicate treatment start date. Kaplan–Meier analyses of the mice are shown.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Mutagenesis, Clone Assay, Incubation, Control, Western Blot, Immunohistochemistry, Staining, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Immunoprecipitation