Direct Observation of Endocytosis Dynamics of Anti-ErbB Modified Single Nanocargoes

The ErbB receptor family, including the epidermal growth factor receptor (EGFR) and ErbB2/3/4, regulate cell proliferation, differentiation, apoptosis, motility, etc., and their abnormalities can cause cancer and other diseases. Ligand-induced endocytosis of ErbB receptors is the key to various cancer treatment strategies, and different techniques have been developed to study this important process. Among them, single particle tracking (SPT) can reveal the spatiotemporal heterogeneity of ErbB receptors on the live cell membrane and has been used to characterize the EGFR dimerization process. Herein, we studied the endocytosis dynamics of two different ErbB receptors using dark-field microscopy. With anti-ErbB modified plasmonic gold nanorods (AuNRs) as probes, we compared the trajectories of individual anti-EGFR AuNRs (cAuNRs) and anti-ErbB AuNRs (tAuNRs) interacting with MCF-7 cells in situ in real time. The results revealed that the internalization rate of cAuNRs was faster than that of tAuNRs. Detailed SPT analysis suggests that cAuNRs enter cells through EGFR endocytosis pathway, and multiple intracellular transport modes sort the cAuNRs away from the transmembrane site. In contrast, the endocytosis resistance of ErbB2 slows down the cellular uptake rate of tAuNRs and causes some tAuNRs-ErbB2 complexes to be confined on the membrane with “circular” and “rolling circle” motions for a much longer time. Our results provide insights into the endocytosis process of the ErbB receptor family at the nanometer scale and could be potentially useful to develop cancer treatment strategies

Direct Observation of Endocytosis Dynamics of Anti-ErbB Modified Single Nanocargoes

The ErbB receptor family, including the epidermal growth factor receptor (EGFR) and ErbB2/3/4, regulate cell proliferation, differentiation, apoptosis, motility, etc., and their abnormalities can cause cancer and other diseases. Ligand-induced endocytosis of ErbB receptors is the key to various cancer treatment strategies, and different techniques have been developed to study this important process. Among them, single particle tracking (SPT) can reveal the spatiotemporal heterogeneity of ErbB receptors on the live cell membrane and has been used to characterize the EGFR dimerization process. Herein, we studied the endocytosis dynamics of two different ErbB receptors using dark-field microscopy. With anti-ErbB modified plasmonic gold nanorods (AuNRs) as probes, we compared the trajectories of individual anti-EGFR AuNRs (cAuNRs) and anti-ErbB AuNRs (tAuNRs) interacting with MCF-7 cells in situ in real time. The results revealed that the internalization rate of cAuNRs was faster than that of tAuNRs. Detailed SPT analysis suggests that cAuNRs enter cells through EGFR endocytosis pathway, and multiple intracellular transport modes sort the cAuNRs away from the transmembrane site. In contrast, the endocytosis resistance of ErbB2 slows down the cellular uptake rate of tAuNRs and causes some tAuNRs-ErbB2 complexes to be confined on the membrane with “circular” and “rolling circle” motions for a much longer time. Our results provide insights into the endocytosis process of the ErbB receptor family at the nanometer scale and could be potentially useful to develop cancer treatment strategies

Direct Observation of Endocytosis Dynamics of Anti-ErbB Modified Single Nanocargoes

The ErbB receptor family, including the epidermal growth factor receptor (EGFR) and ErbB2/3/4, regulate cell proliferation, differentiation, apoptosis, motility, etc., and their abnormalities can cause cancer and other diseases. Ligand-induced endocytosis of ErbB receptors is the key to various cancer treatment strategies, and different techniques have been developed to study this important process. Among them, single particle tracking (SPT) can reveal the spatiotemporal heterogeneity of ErbB receptors on the live cell membrane and has been used to characterize the EGFR dimerization process. Herein, we studied the endocytosis dynamics of two different ErbB receptors using dark-field microscopy. With anti-ErbB modified plasmonic gold nanorods (AuNRs) as probes, we compared the trajectories of individual anti-EGFR AuNRs (cAuNRs) and anti-ErbB AuNRs (tAuNRs) interacting with MCF-7 cells in situ in real time. The results revealed that the internalization rate of cAuNRs was faster than that of tAuNRs. Detailed SPT analysis suggests that cAuNRs enter cells through EGFR endocytosis pathway, and multiple intracellular transport modes sort the cAuNRs away from the transmembrane site. In contrast, the endocytosis resistance of ErbB2 slows down the cellular uptake rate of tAuNRs and causes some tAuNRs-ErbB2 complexes to be confined on the membrane with “circular” and “rolling circle” motions for a much longer time. Our results provide insights into the endocytosis process of the ErbB receptor family at the nanometer scale and could be potentially useful to develop cancer treatment strategies

Direct Observation of Endocytosis Dynamics of Anti-ErbB Modified Single Nanocargoes

The ErbB receptor family, including the epidermal growth factor receptor (EGFR) and ErbB2/3/4, regulate cell proliferation, differentiation, apoptosis, motility, etc., and their abnormalities can cause cancer and other diseases. Ligand-induced endocytosis of ErbB receptors is the key to various cancer treatment strategies, and different techniques have been developed to study this important process. Among them, single particle tracking (SPT) can reveal the spatiotemporal heterogeneity of ErbB receptors on the live cell membrane and has been used to characterize the EGFR dimerization process. Herein, we studied the endocytosis dynamics of two different ErbB receptors using dark-field microscopy. With anti-ErbB modified plasmonic gold nanorods (AuNRs) as probes, we compared the trajectories of individual anti-EGFR AuNRs (cAuNRs) and anti-ErbB AuNRs (tAuNRs) interacting with MCF-7 cells in situ in real time. The results revealed that the internalization rate of cAuNRs was faster than that of tAuNRs. Detailed SPT analysis suggests that cAuNRs enter cells through EGFR endocytosis pathway, and multiple intracellular transport modes sort the cAuNRs away from the transmembrane site. In contrast, the endocytosis resistance of ErbB2 slows down the cellular uptake rate of tAuNRs and causes some tAuNRs-ErbB2 complexes to be confined on the membrane with “circular” and “rolling circle” motions for a much longer time. Our results provide insights into the endocytosis process of the ErbB receptor family at the nanometer scale and could be potentially useful to develop cancer treatment strategies

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells-0

(sample from patient 1), CESS, K562, HL60, and RPMI 8226 and SGH-MM5 MM cell lines using a denaturing and reducing gel-loading buffer (95°C for 10 mins) (A). Cell extracts were also prepared using conventional methods from previously frozen and stored human PB T cells (samples from patients 2 and 3), K562 CML, and HL60, and RPMI 8226 and SGH-MM5 MM cell lines (B). Cell lysates (20.0 μg/sample) were resolved on a 12.5% SDS-PAGE gel, transferred onto PVDF membranes, and probed with S10B1 anti-Ku86 mAb, which recognizes the N-terminus of Ku86. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Experiments were performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells"</p><p>http://www.cancerci.com/content/8/1/4</p><p>Cancer Cell International 2008;8():4-4.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2386117.</p><p></p

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells-5

(sample from patient 1), CESS, K562, HL60, and RPMI 8226 and SGH-MM5 MM cell lines using a denaturing and reducing gel-loading buffer (95°C for 10 mins) (A). Cell extracts were also prepared using conventional methods from previously frozen and stored human PB T cells (samples from patients 2 and 3), K562 CML, and HL60, and RPMI 8226 and SGH-MM5 MM cell lines (B). Cell lysates (20.0 μg/sample) were resolved on a 12.5% SDS-PAGE gel, transferred onto PVDF membranes, and probed with S10B1 anti-Ku86 mAb, which recognizes the N-terminus of Ku86. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Experiments were performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells"</p><p>http://www.cancerci.com/content/8/1/4</p><p>Cancer Cell International 2008;8():4-4.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2386117.</p><p></p

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells-2

Thout H (500 U/reaction) for 2 hrs or 24 hrs (A); or with varying amounts (0, 1, 2, 3, 4 or 5 μL) of H (500 U/μL) for 20 hrs (B; positive control); and then resolved in a 12.5% SDS-PAGE gel, and immunoblotted using S10B1 anti-Ku86 (A), or anti-heavy chain of the human MHC class I protein mAbs (B). Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Experiments were performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells"</p><p>http://www.cancerci.com/content/8/1/4</p><p>Cancer Cell International 2008;8():4-4.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2386117.</p><p></p

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells-3

for 18 hrs with either 1× or 2× Complete™ protease inhibitor tablets (lanes 2 and 3); and then washed and checked for viability using trypan blue exclusion assay. Mock experiments in which cell lines were incubated for 18 hrs with media alone (lane 1) served as negative controls. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. The RPMI 8226 MM cell line s (B) was also incubated for 24 hrs with 2× Complete™ protease inhibitor tablets (lane 2), antipain (2.0 μg/mL final concentration) plus leupeptin (2.0 μg/mL final concentration) (lane 3), or aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) (lane 4) and analyzed in the same way as above. Mock experiments in which cell lines were incubated for 24 hrs with media alone (lane 1) again served as negative controls. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. All experiments were performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells"</p><p>http://www.cancerci.com/content/8/1/4</p><p>Cancer Cell International 2008;8():4-4.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2386117.</p><p></p

Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells

Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells-4

.5 μg/sample) by trypsin (0.065 μg of Trypsin Gold/reaction up to 20 mins) was then performed using a limited proteolysis protocol from the manufacturer. The reactions were stopped by rapid cooling on ice; and the products of trypsin digestion were resolved by SDS PAGE and immunoblotted S10B1 anti-Ku86 mAb. The effect of protease inhibitors aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) on trypsin digestion was also determined on a larger sample of rhKu86 (13.0 μg/sample). Trypsin digestion (0.065 μg of Trypsin Gold/reaction for 1 min) of rhKu86 was performed as above either without (lane 1) or with (lane 2) protease inhibitors (B).<p><b>Copyright information:</b></p><p>Taken from "Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells"</p><p>http://www.cancerci.com/content/8/1/4</p><p>Cancer Cell International 2008;8():4-4.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2386117.</p><p></p