Journal: Molecular cancer therapeutics

Article Title: Development of a MUC16-Targeted Near-Infrared Fluorescent Antibody Conjugate for Intraoperative Imaging of Pancreatic Cancer

doi: 10.1158/1535-7163.MCT-20-0033

Figure Lengend Snippet: Synthesis and in vitro characterization of antibody conjugates. (A) Schematic of IRDye800 NHS Ester conjugation to AR9.6 and IgG. (B) Representative absorbance and emission spectra from both antibody conjugates. (C) Western blot of MUC16 expression in human pancreatic cancer cell lines. (D) Fluorescent western blot confirming binding of AR9.6-IRDye800, and lack of binding in IgG control. (E) Immunofluorescence of antibody conjugate binding. Images acquired at 400X magnification. Scale bar = 20 μm.

Article Snippet: After washing, the membrane was incubated with goat anti-mouse IRDye800 (1:20,000, LI-COR Biosciences, 926–33210) in 5% blotting grade blocker in TBST, washed, and imaged on Odyssey CLx (LI-COR Biosciences).

Techniques: In Vitro, Conjugation Assay, Western Blot, Expressing, Binding Assay, Immunofluorescence

Journal: Molecular cancer therapeutics

Article Title: Development of a MUC16-Targeted Near-Infrared Fluorescent Antibody Conjugate for Intraoperative Imaging of Pancreatic Cancer

doi: 10.1158/1535-7163.MCT-20-0033

Figure Lengend Snippet: Tumor accumulation of fluorescent antibody conjugates. (A) Representative images from the biodistribution of the fluorescent conjugates AR9.6-IRDye800, IgG-IRDye800, and unconjugated IRDye800, from 4 −144 h (N = 4). Normalized arbitrary units (AU) depicted on the color bar is representative of all images. (B) Signal to noise ratios and tumor to background ratios of AR9.6-IRDye800, IgG-IRDye800, and unconjugated IRDye800 over 144 h.

Article Snippet: After washing, the membrane was incubated with goat anti-mouse IRDye800 (1:20,000, LI-COR Biosciences, 926–33210) in 5% blotting grade blocker in TBST, washed, and imaged on Odyssey CLx (LI-COR Biosciences).

Techniques:

Journal: Molecular cancer therapeutics

Article Title: Development of a MUC16-Targeted Near-Infrared Fluorescent Antibody Conjugate for Intraoperative Imaging of Pancreatic Cancer

doi: 10.1158/1535-7163.MCT-20-0033

Figure Lengend Snippet: Fluorescence-Guided Surgery in orthotopic pancreatic cancer. Representative Fluobeam images of FGS with (A) AR9.6-IRDye800 and (B) IgG-IRDye800. (C) Calculated TBR (tumor signal/ adjacent normal pancreas or adjacent peritoneal tissue) in tumors (p = 0.0010). (D) Representative images of anterior and posterior pancreas imaging with AR9.6-IRDye800 using the Lab Flare RP1.

Article Snippet: After washing, the membrane was incubated with goat anti-mouse IRDye800 (1:20,000, LI-COR Biosciences, 926–33210) in 5% blotting grade blocker in TBST, washed, and imaged on Odyssey CLx (LI-COR Biosciences).

Techniques: Fluorescence, Imaging

Journal: Molecular cancer therapeutics

Article Title: Development of a MUC16-Targeted Near-Infrared Fluorescent Antibody Conjugate for Intraoperative Imaging of Pancreatic Cancer

doi: 10.1158/1535-7163.MCT-20-0033

Figure Lengend Snippet: Biodistribution and signal of AR9.6-IRDye800 and IgG-IRDye800 in an orthotopic xenograft model. (A) Biodistribution of AR9.6-IRDye800 and IgG-IRDye800 at 144 h post-injection in necropsied organs. (N=4)) 1=Tumor and Unaffected Pancreas, 2=Heart, 3=Lung, 4=Liver, 5=Spleen, 6=Kidney, 7=Stomach, 8=Large Intestine, 9=Small Intestine, 10=Muscle, 11=Bone. (B) Mean fluorescent signal across necropsied organs. (p = 0.00095) (C) Calculated SNR (tumor signal/ standard deviation of background) in tumors (p = 0.0010).

Article Snippet: After washing, the membrane was incubated with goat anti-mouse IRDye800 (1:20,000, LI-COR Biosciences, 926–33210) in 5% blotting grade blocker in TBST, washed, and imaged on Odyssey CLx (LI-COR Biosciences).

Techniques: Injection, Standard Deviation

Journal: Molecular cancer therapeutics

Article Title: Development of a MUC16-Targeted Near-Infrared Fluorescent Antibody Conjugate for Intraoperative Imaging of Pancreatic Cancer

doi: 10.1158/1535-7163.MCT-20-0033

Figure Lengend Snippet: Spectral analysis of tumors and fluorescent histology. (A) Pearl® Trilogy imaging, H&E staining, and acquired spectra for AR9.6-IRDye800 tumor. The red box denotes the area depicted by the H&E image, and the black arrow denotes tumor. (B) Pearl® Trilogy imaging, H&E staining and acquired spectra for IgG-IRDye800 tumor. (C) Fluorescence microscopy in tumors. Images acquired at 200X magnification. Scale bars = 50 μm.

Article Snippet: After washing, the membrane was incubated with goat anti-mouse IRDye800 (1:20,000, LI-COR Biosciences, 926–33210) in 5% blotting grade blocker in TBST, washed, and imaged on Odyssey CLx (LI-COR Biosciences).

Techniques: Imaging, Staining, Fluorescence, Microscopy

Journal: Autophagy

Article Title: Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

doi: 10.1080/15548627.2016.1183844

Figure Lengend Snippet: Cocaine-mediated activation of autophagy in human A172 astrocytoma cells and primary human astrocytes. (A to D) Representative western blots showing the time-dependent activation of autophagy marker proteins, such as (A) BECN1, (B) ATG5, (C) MAP1LC3B-II and (D) SQSTM1, in overnight serum-starved human A172 astrocytoma cells that were treated with 10 µM cocaine for the indicated time durations. (E to H) Representative western blots showing the time-dependent activation of autophagy marker proteins, such as (E) BECN1, (F) ATG5, (G) MAP1LC3B-II and (H) SQSTM1, in overnight serum-starved primary human astrocytes that were treated with 10 µM cocaine for the indicated time durations. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from 3 independent experiments. One-way ANOVA followed by post hoc test least significant difference was used to determine the statistical significance. *, P < 0.05 vs control group.

Article Snippet: Antibodies were obtained from the following sources: BECN1 (sc-11427), ATG5 (sc-33210), p-EIF2AK3 (sc-32577), EIF2AK3 (sc-13073), ERN1 (sc-20790), ATF6 (sc-22799), goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology; EIF2S1 (5324), p-EIF2S1 (3398), and DDIT3 (2895) were from Cell Signaling Technology; MAP1LC3B (NB100-2220) was from Novus Biological Company; HSPA5 (610979) was from BD Biosciences and SQSTM1 (PM045) was from MBL International.

Techniques: Activation Assay, Western Blot, Marker

Journal: Autophagy

Article Title: Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

doi: 10.1080/15548627.2016.1183844

Figure Lengend Snippet: Cocaine-mediated autophagy involved upstream activation of ER stress in human A172 astrocytoma cells. (A to D) Representative western blots showing the protein levels of autophagy markers, such as (A) BECN1, (B) ATG5, (C) MAP1LC3B-II and (D) SQSTM1, in human A172 astrocytoma cells pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 µM cocaine for 12 h. (E and F) Representative western blots showing the protein levels of ER stress markers, such as (E) HSPA5 and (F) p-EIF2S1:EIF2S1, in human A172 astrocytoma cells pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 µM cocaine for 12 h. (G) Representative western blots showing the protein levels of an autophagy marker, such as BECN1, in human A172 astrocytoma cells transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. (H) Representative western blots showing the protein levels of ER stress marker such as HSPA5 in human A172 astrocytoma cells transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. *, P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

Article Snippet: Antibodies were obtained from the following sources: BECN1 (sc-11427), ATG5 (sc-33210), p-EIF2AK3 (sc-32577), EIF2AK3 (sc-13073), ERN1 (sc-20790), ATF6 (sc-22799), goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology; EIF2S1 (5324), p-EIF2S1 (3398), and DDIT3 (2895) were from Cell Signaling Technology; MAP1LC3B (NB100-2220) was from Novus Biological Company; HSPA5 (610979) was from BD Biosciences and SQSTM1 (PM045) was from MBL International.

Techniques: Activation Assay, Western Blot, Marker, Transfection

Journal: Autophagy

Article Title: Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

doi: 10.1080/15548627.2016.1183844

Figure Lengend Snippet: Cocaine-mediated autophagy involved upstream activation of ER stress in primary human astrocytes. (A to D) Representative western blots showing the protein levels of autophagy markers, such as (A) BECN1, (B) ATG5, (C) MAP1LC3B-II and (D) SQSTM1, in primary human astrocytes pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 µM cocaine for 12 h. (E and F) Representative western blots showing the protein levels of ER stress markers, such as (E) HSPA5 and (F) p-EIF2S1:EIF2S1, in primary human astrocytes pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 µM cocaine for 12 h. (G) Representative western blots showing the protein levels of an autophagy marker, such as BECN1, in primary human astrocytes transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. (H) Representative western blots showing the protein levels of ER stress marker such as HSPA5 in primary human astrocytes transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. *, P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

Article Snippet: Antibodies were obtained from the following sources: BECN1 (sc-11427), ATG5 (sc-33210), p-EIF2AK3 (sc-32577), EIF2AK3 (sc-13073), ERN1 (sc-20790), ATF6 (sc-22799), goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology; EIF2S1 (5324), p-EIF2S1 (3398), and DDIT3 (2895) were from Cell Signaling Technology; MAP1LC3B (NB100-2220) was from Novus Biological Company; HSPA5 (610979) was from BD Biosciences and SQSTM1 (PM045) was from MBL International.

Techniques: Activation Assay, Western Blot, Marker, Transfection

Journal: Autophagy

Article Title: Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

doi: 10.1080/15548627.2016.1183844

Figure Lengend Snippet: Cocaine-mediated autophagy involved upstream activation of ER stress in human A172 astrocytoma cells. (A and B) Representative western blots showing the protein levels of ER stress markers, such as (A) HSPA5, and (B) p-EIF2S1:EIF2S1, in human A172 astrocytoma cells pretreated with the ER stress inhibitors, such as 10 µM salubrinal or 50 µM 4-PBA, for 1 h following treatment with 10 µM cocaine for 12 h. (C to F) Representative western blots showing the protein levels of autophagy markers, such as (C) BECN1, (D) ATG5, (E) MAP1LC3B-II and (F) SQSTM1, in human A172 astrocytoma cells pretreated with 10 µM salubrinal or 50 µM 4-PBA for 1 h following treatment with 10 µM cocaine for 12 h. (G) Representative western blots showing the protein levels of an ER stress marker, HSPA5, in human A172 astrocytoma cells transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. (H) Representative western blots showing the protein levels of autophagy marker, BECN1 in human A172 astrocytoma cells transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. *, P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

Article Snippet: Antibodies were obtained from the following sources: BECN1 (sc-11427), ATG5 (sc-33210), p-EIF2AK3 (sc-32577), EIF2AK3 (sc-13073), ERN1 (sc-20790), ATF6 (sc-22799), goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology; EIF2S1 (5324), p-EIF2S1 (3398), and DDIT3 (2895) were from Cell Signaling Technology; MAP1LC3B (NB100-2220) was from Novus Biological Company; HSPA5 (610979) was from BD Biosciences and SQSTM1 (PM045) was from MBL International.

Techniques: Activation Assay, Western Blot, Marker, Transfection

Journal: Autophagy

Article Title: Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

doi: 10.1080/15548627.2016.1183844

Figure Lengend Snippet: Cocaine-mediated autophagy involved upstream activation of ER stress in primary human astrocytes. (A and B) Representative western blots showing the protein levels of ER stress markers, such as (A) HSPA5, and (B) p-EIF2S1:EIF2S1, in primary human astrocytes pretreated with the ER stress inhibitors, such as 10 µM salubrinal or 50 µM 4-PBA, for 1 h following treatment with 10 µM cocaine for 12 h. (C to F) Representative western blots showing the protein levels of autophagy markers, such as (C) BECN1, (D) ATG5, (E) MAP1LC3B-II and (F) SQSTM1, in primary human astrocytes pretreated with 10 µM salubrinal or 50 µM 4-PBA for 1 h following treatment with 10 µM cocaine for 12 h. (G) Representative western blots showing the protein levels of an ER stress marker, HSPA5, in primary human astrocytes transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. (H) Representative western blots showing the protein levels of autophagy marker, BECN1 in primary human astrocytes transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 µM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. *, P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

Article Snippet: Antibodies were obtained from the following sources: BECN1 (sc-11427), ATG5 (sc-33210), p-EIF2AK3 (sc-32577), EIF2AK3 (sc-13073), ERN1 (sc-20790), ATF6 (sc-22799), goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology; EIF2S1 (5324), p-EIF2S1 (3398), and DDIT3 (2895) were from Cell Signaling Technology; MAP1LC3B (NB100-2220) was from Novus Biological Company; HSPA5 (610979) was from BD Biosciences and SQSTM1 (PM045) was from MBL International.

Techniques: Activation Assay, Western Blot, Marker, Transfection