Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Significant mRNA overexpression of t-DARPP and ERBB2 in adenocarcinomas of the esophagus and stomach (141 tumors and 51 normal tissue samples) (p<0.001). B) Spearman’s correlation coefficient and correlation test where the cutoff gene expression is ≥log(5,2)=2.32, show that t-DARPP and ERBB2 overexpression levels are significantly correlated in tumors (r=0.58, p=0.003). C) The multivariate regression model analysis indicates that tumor stage has a significant effect on t-DARPP mRNA gene expression levels (p=0.02). D) Left panel, cell viability of OE19 and OE33 cells in response to trastuzumab treatment was evaluated by Trypan blue staining. OE19 cells were two-fold more sensitive to trastuzumab than OE33 cells (p<0.001). Right panel, Western blot analysis demonstrates higher protein expression of ERBB2 in OE19 cells than OE33 cells. In contrast, t-DARPP expression was undetectable in OE19 cells but highly expressed in OE33 cells.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Over Expression, Expressing, Staining, Western Blot

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) ERBB2 protein stability in OE19 cells stably expressing t-DARPP or pcDNA3 empty vector was evaluated by Western blot analysis after treatment with 80 μg/ml CHX to block new protein synthesis for the indicated times. The protein degradation data indicate that t-DARPP expression extended the protein half-life of ERBB2 from 30.8 h to 42.2 h relative to control (lower panel). B) ERBB2 protein stability in parental and trastuzumab resistant OE19 cells was assessed by Western blot analysis after treatment with CHX (80 μg/ml) for the indicated times. The protein degradation data show that endogenous t-DARPP expression in resistant cells was associated with increased ERBB2 protein half-life (60.8 h) relative to parental cells (31.3 h) (lower panel). C) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The data indicate that transient expression of t-DARPP increased p-ERBB2(Y1248) and p-AKT(S473) basal protein levels, and blocked trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. D) Western blot analysis of p-ERBB2 (Y1248), ERBB2, p-AKT (S473), AKT, and t-DARPP proteins in OE19 cells stably expressing t-DARPP or pcDNA3 vector after treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results show that stable expression of t-DARPP increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and inhibited trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. E) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in parental or trastuzumab resistant OE19 cells following treatment with vehicle or trastuzumab (20 μg/ml) for 24 h. The results indicate that endogenous t-DARPP expression was associated with increased basal levels of p-ERBB2(Y1248) and p-AKT(S473), and suppression of trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Blocking Assay, Infection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of p-ERBB2(Y1248), ERBB2, p-AKT(S473), AKT, and t-DARPP proteins in OE33 cells transfected with control siRNA or t-DARPP siRNA and treated with vehicle or trastuzumab (20 μg/ml) for 48 h. The data indicate that knockdown of endogenous t-DARPP increased trastuzumab-dependent dephosphorylation of ERBB2 and AKT proteins. B) Cell viability of OE33 cells transfected with control siRNA or t-DARPP siRNA in response to treatment with vehicle or trastuzumab (20 μg/ml) for 48 h, was evaluated by CellTiter-Glo Luminescent CellViability Assay. The results revealed that knockdown of endogenous t-DARPP with treatment induced a significant decrease in cell survival (p<0.01).

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Transfection, De-Phosphorylation Assay

Journal: Cancer research

Article Title: Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

doi: 10.1158/0008-5472.CAN-12-1119

Figure Lengend Snippet: A) Western blot analysis of co-immunoprecipitated exogenous t-DARPP and endogenous ERBB2 proteins with M2-flag or trastuzumab antibodies in OE19 cells infected with t-DARPP-flag adenovirus (10 MOI). The data demonstrate protein association of ERBB2 with t-DARPP. B) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses. Pulled-down ERBB2 band intensity was depicted as a ratio relative to input ERBB2 protein. The results show that exogenous t-DARPP expression blocked binding of trastuzumab to ERBB2 receptor relative to control. C) Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in parental or trastuzumab resistant OE19 cells. The band intensity of immunoprecipitated ERBB2 protein was shown as a ratio relative to input ERBB2. The data indicate that endogenous t-DARPP expression in trastuzumab-resistant cells was associated with a significant decrease in trastuzumab/ERBB2 protein interaction relative to control.

Article Snippet: DARPP-32 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and P-ERBB2(Y1248) antibody was obtained from Abcam (San Francisco, CA).

Techniques: Western Blot, Immunoprecipitation, Infection, Expressing, Binding Assay

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Brusatol in combination with lapatinib synergistically inhibited the growth of HER2-overexpressed SK-BR-3, SK-OV-3 and AU565 cells (A) SK-BR-3, SK-OV-3 and AU565 cell lines were treated with brusatol, lapatinib or brusatol plus lapatinib in a dose range for 48 h. CCK-8 assays were used to measure the cell viability. Points, mean of 3 independent CCK-8 assays; Bars, SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (B) The synergistic effect of lapatinib in combination with brusatol was evaluated on the growth of SK-BR-3, SK-OV-3 and AU565 cell line. Combination index (CI) values were calculated using the Chou-Talalay method. Drug synergy, addition, and antagonism are defined by CI values less than 1.0, equal to 1.0, or greater than 1.0, respectively.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: CCK-8 Assay

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Lapatinib plus brusatol abrogate the activation of Nrf2/HO-1 and EGFR/HER2-AKT/ERK1/2 pathways (A and B) SK-BR-3 and SK-OV-3 cells were treated with lapatinib or brusatol alone, or their combination for 24 h. The changes in Nrf2/HO-1 and EGFR/HER2-AKT/ERK1/2 signaling pathways were monitored by Western Blotting (C and D) Densitometric analysis was performed on the Western Blotting. The levels of Nrf2, HO-1, p-HER2, p-EGFR, p-AKT and p-ERK1/2 were quantified by using the software Image J. The data are expressed as the mean ± SD of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. The original blots were provided in supplementary data as Supplementary Figure S4.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Software

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Nrf2 knockdown repressed the activation of HER2 signaling pathway and sensitizes SK-OV-3 cells to lapatinib treatment (A) Effect of Nrf2 knockdown on the expression of HO-1, p-HER2, p-AKT, and p-ERK1/2 were determined after treatment with Nrf2 siRNA or scramble siRNA for 36 h (B) Effect of Nrf2 knockdown on the sensitivity to lapatinib. Cell viability was examined after lapatinib treatment for 36 h in Nrf2 siRNA or scramble siRNA-transfected cells. Cells were transfected with Nrf2 siRNA or scramble siRNA using Lipofectamine 3000 (Invitrogen) according to the supplier's instruction. Data show the mean ± SD (three independent experiments). ∗∗∗ p < 0.001. The original blots were provided in supplementary data as Supplementary Figure S5.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Knockdown, Activation Assay, Expressing, Transfection

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Proposed model of the molecular basis of synergistic interaction between lapatinb and brusatol. Brusatol, in combination with lapatinib, may exert synergistic effects in two ways: (a) combination therapy inhibits the phosphorylation of HER receptors including EGFR and HER2, limiting the activation of their downstream pathways including PI3K-AKT signaling and Ras/Raf/MAPK signaling. (b) combination therapy modulates cell redox homeostasis by decreasing Nrf2 level and preventing the accumulation of Nrf2 in the nucleus, interfering its binding to small Maf oncogene family proteins (Mafs) and antioxidant response elements (AREs) complex, causing the inhibition of antioxidant genes such as heme oxygenase 1 (HO-1) and superoxide dismutase (SOD), thereby resulting in ROS accumulation and cell death.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Phospho-proteomics, Activation Assay, Binding Assay, Inhibition

Journal: Orphanet Journal of Rare Diseases

Article Title: Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

doi: 10.1186/s13023-019-1241-z

Figure Lengend Snippet: The c.1253 T > C (p.I418T) mutation has no effect on ERBB3 expression and interaction with ERBB2, whereas the c.3182dupA (p.N1061Kfs*16) mutation produces a novel truncated protein. a Western blotting results using anti-Myc antibody to detect ERBB3 in lysates of HEK293T cells transfected with 2 μg empty vector (EV) or WT, c.1253 T > C (M1), or c.3182dupA (M2) plasmids. b Results of co-immunoprecipitation to detect the interaction between ERBB2 and WT or I418T (M1) mutant ERBB3

Article Snippet: Antibodies against ERBB2 (#2242), phosphorylated (p-)ERBB2 (Tyr1248) (#2247), ERBB3 (C-terminus) (#12708), p-ERBB3 (Tyr1289) (#4791), ERK (#4695), p-ERK (#4370), AKT (#4685), p-AKT (#4060), GFP (#2555), Myc (#2272), and GAPDH (#5174) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Expressing, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Orphanet Journal of Rare Diseases

Article Title: Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

doi: 10.1186/s13023-019-1241-z

Figure Lengend Snippet: ERBB3 variants lack the capacity to activate PI3K/AKT and ERK signaling pathways. a Immunoblot analysis was performed using indicated antibodies to determine the effects of WT or mutant ERBB3 on PI3K/AKT and ERK pathway activation. To induce protein phosphorylation, HEK293T cells were treated with 10 ng/ml NRG-1β for 30 min after transfection with empty vector (EV), WT, M1, M2, or M3 (V104 L) plasmids. b–e Quantitative analysis of p-ERK, p-AKT, p-ERBB2, and p–ERBB3 expression. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT

Article Snippet: Antibodies against ERBB2 (#2242), phosphorylated (p-)ERBB2 (Tyr1248) (#2247), ERBB3 (C-terminus) (#12708), p-ERBB3 (Tyr1289) (#4791), ERK (#4695), p-ERK (#4370), AKT (#4685), p-AKT (#4060), GFP (#2555), Myc (#2272), and GAPDH (#5174) were purchased from Cell Signaling Technology.

Techniques: Protein-Protein interactions, Western Blot, Mutagenesis, Activation Assay, Phospho-proteomics, Transfection, Plasmid Preparation, Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: Irreversible tyrosine kinase inhibitors induce the endocytosis and downregulation of ErbB2

doi: 10.1016/j.bbrep.2023.101436

Figure Lengend Snippet: Irreversible ErbB2 TKIs induce the downregulation of ErbB2. UMSCC1 cells were treated with vehicle control (DMSO, 1:1000) or four different TKIs at the concentrations as indicated for 6 h, and then the indicated protein levels were detected by Western blot (A) and the ErbB2 levels were quantified (B). UMSCC1 cells were treated with vehicle control (DMSO 1:1000), Dacomitinib (1 μM), or Afatinib (1 μM) for 6 h and the ErbB2 mRNA levels were quantified by Real-time PCR (C). UMSCC22B cells were treated with vehicle control (DMSO 1:1000) or different TKIs as indicated, and then the indicated protein levels were detected by Western blot (D) and the ErbB2 levels were quantified (E). MDA-MB-453 cells were treated with vehicle control (DMSO 1:1000), or different TKIs as indicated, and then the indicated protein levels were detected by Western blot (F) and the ErbB2 levels were quantified (G). Error bars indicate mean ± SEM from three independent experiments. * P < 0.01, ** P < 0.001, *** P < 0.0001.

Article Snippet: Antibodies to EGFR, phosphor-EGFR (Tyr1068), phosphor-ErbB2 (Tyr1196), total AKT, phosphor-AKT, total ERK1/2, phosphor-ERK1/2, LAMP1, and Rab7 were from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Biochemistry and Biophysics Reports

Article Title: Irreversible tyrosine kinase inhibitors induce the endocytosis and downregulation of ErbB2

doi: 10.1016/j.bbrep.2023.101436

Figure Lengend Snippet: Irreversible TKI-induced ErbB2 downregulation is not dependent on lysosome or proteasome. UMSCC1 cells were treated with vehicle control (DMSO 1:1000), Dacomitinib (1 μM), combination of Dacomitinib with Chloroquine (50 μM), Velcade (1 μM), or Pr-171 (1 μM) as indicated for 6 h, and then the ErbB2 and p -ErbB2 protein levels were detected by Western blot (A) and the ErbB2 levels were quantified (B). UMSCC1 cells were treated with vehicle control (DMSO 1:1000), Dacomitinib (1 μM), ALLN (130 μM) and their combination as indicated for 6 h and then the protein levels of ErbB2 and p -ErbB2 were measured by Western blot (C) and the ErbB2 levels were quantified (D). Error bars indicate mean ± SEM from three independent experiments. *** P < 0.0001.

Article Snippet: Antibodies to EGFR, phosphor-EGFR (Tyr1068), phosphor-ErbB2 (Tyr1196), total AKT, phosphor-AKT, total ERK1/2, phosphor-ERK1/2, LAMP1, and Rab7 were from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Irreversible tyrosine kinase inhibitors induce the endocytosis and downregulation of ErbB2

doi: 10.1016/j.bbrep.2023.101436

Figure Lengend Snippet: Irreversible TKI stimulates the endocytosis and accumulation of ErbB2 at endosomes. MDA-MB-453 cells were treated with vehicle control (DMSO 1:1000), Dacomitinib (1 μM), or Lapatinib (1 μM) for 6 h, and then the subcellular location of ErbB2 and LAMP1 were detected by confocal microscopy (A). ErbB2 fluorescence intensity on plasma membrane was quantified (B). Colocalization of ErbB2 with LAMP1 was quantified (C). The subcellular location of ErbB2 and Rab7 were detected by confocal microscopy (D). Colocalization of ErbB2 with Rab7 was quantified (E). Insets show high magnifications of the corresponding framed areas. Scale bars 10 μm. Error bars indicate mean ± SEM from three independent experiments. *** P < 0.0001.

Article Snippet: Antibodies to EGFR, phosphor-EGFR (Tyr1068), phosphor-ErbB2 (Tyr1196), total AKT, phosphor-AKT, total ERK1/2, phosphor-ERK1/2, LAMP1, and Rab7 were from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Confocal Microscopy, Fluorescence, Clinical Proteomics, Membrane

Journal: Biochemistry and Biophysics Reports

Article Title: Irreversible tyrosine kinase inhibitors induce the endocytosis and downregulation of ErbB2

doi: 10.1016/j.bbrep.2023.101436

Figure Lengend Snippet: Irreversible TKI-induced ErbB2 downregulation requires the dynamin-dependent but clathrin-independent endocytosis. MDA-MB-453 cells were treated with vehicle control (DMSO 1:1000), Dacomitinib (1 μM), and the combination of Dacomitinib (1 μM) with Dyngo-4a (30 μM) or with PitStop2 (30 μM) as indicated for 6 h, and then the subcellular location of ErbB2 and LAMP1 were detected by confocal microscopy (A). ErbB2 fluorescence intensity on plasma membrane was quantified (B). Colocalization of ErbB2 with LAMP1 was quantified (C). The subcellular location of ErbB2 and Rab7 were detected by confocal microscopy (D). Colocalization of ErbB2 with Rab7 was quantified (E). MDA-MB-453 cells were treated with vehicle control (DMSO 1:1000), Dacomitinib (1 μM), Dyngo-4a (30 μM), Pitstop2 (30 μM) and their combination as indicated for 6 h, and then ErbB2 protein expression levels were measured by Western blot (F) and quantified (G). Insets show high magnifications of the corresponding framed areas. Scale bars 10 μm. Error bars indicate mean ± SEM from three independent experiments. *** P < 0.0001, n.s.-non-significant.

Article Snippet: Antibodies to EGFR, phosphor-EGFR (Tyr1068), phosphor-ErbB2 (Tyr1196), total AKT, phosphor-AKT, total ERK1/2, phosphor-ERK1/2, LAMP1, and Rab7 were from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Confocal Microscopy, Fluorescence, Clinical Proteomics, Membrane, Expressing, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Traditional Chinese Medicine Tang-Luo-Ning Ameliorates Sciatic Nerve Injuries in Streptozotocin-Induced Diabetic Rats

doi: 10.1155/2013/989670

Figure Lengend Snippet: Effect of TLN and α -LA on Nrg1/ErbB2 downstream signal pathway in sciatic nerve of streptozotocin-induced rats. A representative western blot ((a), (c), (e), and (g)) and the grey value analyses of protein expression ((b), (d), (f), and (h)) in sciatic nerves from different experimental groups. ((a), (b)) Ratio of P-ErbB2 to T-ErbB2 (P/T); ((c), (d)) ratio of P-Erk to T-Erk (P/T); ((e), (f)) ratio of P-Bad (Ser112) to T-Bad (P/T); ((g), (h)) ratio of P-Akt to T-Akt (P/T). Statistical analysis was performed using the ANOVA procedure t test (LSD). Data are expressed as mean ± SD. # P < 0.05 versus CTL group, * P < 0.05 versus STZ group. n = 4 per group. The protein expression of ratio of ErbB2 Erk, Bad (P/T) decreased compared with CTL group ( P < 0.05), while Tang-Luo-Ning and alpha-lipoic acid intervention could increase the ratio above compared with STZ group ( P < 0.05). There were no significant differences of ratio of Akt (P/T) between the four groups. Four groups: CTL group: nonstreptozotocin-induced group, STZ group: streptozotocin-induced diabetic group, TLN group: Tang-Luo-Ning group, and α -LA group: alpha-lipoic acid group. ANOVA: analysis of variance; LSD- t test: least significant difference t test.

Article Snippet: Rabbit polyclonal anti-ErbB2 antibody (dilution: 1 : 4000, Novus, Biologicals, Inc.; Littleton, CO, USA), Rabbit polyclonal anti-Phospho-ErbB2 (Tyr1248) antibody (dilution: 1 : 3000, Novus, Biologicals, Inc.; Littleton, CO, USA), after washing 3 × 10 min with Tris-buffered saline, horseradish peroxidase conjugated secondary antibody (dilution 1 : 1000: P-Bad ser112/ser136, 1 : 2000: other antibodies) was added and incubated for 1 hr with shaking.

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: RUNX1 confers cell proliferative advantage via ErbB2/HER2 signaling. ( a ) Growth curves of NUGC4 and MKN45 cells lentivirally-transduced with control (sh_ Luc ) or with RUNX1 shRNA (sh_ RUNX1 ) in the presence of 3 μM doxycycline (n = 5). ( b ) Apoptotic cell death induced by RUNX1 silencing. Non-depleted and RUNX1 -depleted NUGC4 and MKN45 cells were cultured in the presence of 3 μM doxycycline. Forty-eight hours after treatment, cells were harvested and apoptotic cells (Annexin V + ) were scored by flow cytometric analysis (n = 5). ( c ) RUNX1 expressions were associated with overall survival in the gastric cancer patients. The number of subjects in RUNX1 high (top 10%) and low (bottom 10%) were n = 30 and n = 30. P = 0.048 by log-rank test. Data was retrieved from Gene Expression Omnibus (GEO), accession number; GSE62254. ( d ) Relative densitometric quantification of phospho-RTK array spots in RUNX1 -depleted MKN45 cells compared to the control. Cells were treated with 3 μM doxycycline for 48 hours, then the cells were lysed for the phospho-RTK array. Each receptor was spotted in duplicates (see Supplementary Fig. for the immunoblot image). ( e ) Dephosphorylation of HER2 in RUNX1 -silenced NUGC4 and MKN45 cells. Non-depleted and RUNX1 -depleted cells were treated as in (b). Cell lysates were analyzed by immunoblotting with the indicated antibodies. Data are mean ± SEM values. *P < 0.05, **P < 0.01, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Transduction, Control, shRNA, Cell Culture, Gene Expression, Western Blot, De-Phosphorylation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: Indispensable role of SOS1 in the ErbB2/HER2 signaling cascade. ( a ) Efficacy of shRNAs targeting SOS1 . MKN45 cells were transduced with control (sh_ Luc ) or with SOS1 shRNAs (sh_ SOS1 #1 and #2) and cultured in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). ( b ) SOS1 -depletion-mediated dephosphorylation of HER2. Non-depleted and SOS1 -depleted MKN45 cells were treated as in ( a ). Forty-eight hours after treatment, cell lysates were processed for immunoblotting. ( c ) Growth curves of MKN45 cells transduced with control (sh_ Luc ) or with SOS1 shRNAs (sh_ SOS1 #1 and #2) in the presence of 3 μM doxycycline (n = 5). ( d ) Efficacy of shRNAs targeting HER2 . MKN45 cells were transduced with control (sh_ Luc ) or with HER2 shRNAs (sh_ HER2 #1 and #2) and cultured in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of control vector-transduced cells (n = 3). ( e ) HER2 -depletion-mediated dephosphorylation of HER2. Non-depleted and HER2 -depleted MKN45 cells were treated as in ( d ). Forty-eight hours after treatment, cell lysates were processed for immunoblotting. ( f ) Growth curves of MKN45 cells transduced with control (sh_ Luc ) or with HER2 shRNAs (sh_ HER2 #1 and #2) in the presence of 3 μM doxycycline (n = 5). ( g , h ) Immunoprecipitation assay showed SOS1-HER2 complex in MKN45 cells. Data are mean ± SEM values. *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Transduction, Control, Cell Culture, Quantitative RT-PCR, Plasmid Preparation, De-Phosphorylation Assay, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: Scientific Reports

Article Title: RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

doi: 10.1038/s41598-018-24969-w

Figure Lengend Snippet: RUNX inhibitor Chb-M’ down-regulates ErbB2/HER2 pathway through attenuating SOS1 expression in gastric cancer cells. ( a ) Relative densitometric quantification of phospho-RTK array spots in Chb-M’-treated MKN45 cells compared to the control. Cells were treated with DMSO or 1 μM Chb-M’ for 48 hours, then the cells were lysed for the phospho-RTK array. Each receptor was spotted in duplicates (see Supplementary Fig. for the immunoblot image). ( b ) Down-regulation of SOS1 expression in Chb-M’-treated NUGC4 and MKN45 cells. Cells were treated with the indicated concentrations of Chb-M’. Six hours after treatment, total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized to that of DMSO-treated control cells (n = 3). ( c ) Down-regulation of SOS1 and dephosphorylation of HER2 in Chb-M’-treated NUGC4 and MKN45 cells. Cells were treated as in ( b ). Forty-eight hours after treatment, cell lysates were prepared and subjected to immunoblotting. Data are mean ± SEM values. *P < 0.05, **P < 0.01, n.s.; not significant, by two-tailed Student’s t-test.

Article Snippet: Anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology).

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, De-Phosphorylation Assay, Two Tailed Test

Journal: Cell chemical biology

Article Title: The Advantages of Targeted Protein Degradation over Inhibition: a RTK Case Study

doi: 10.1016/j.chembiol.2017.09.009

Figure Lengend Snippet: Key Resources Table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies EGFR SantaCruz 1005 FLAG Sigma F1804 Tubulin Sigma T9026 HER2 Cell Signaling 2165S p-EGFR (Y1068) Abcam ab40815 p-HER2 (Y1221/1222) Cell Signaling 2243S p-AKT (T308) Cell Signaling 2965S p-ERK 1/2 (T202/204) Cell Signaling 4370 HER3 Cell Signaling 12708 pHER3 (Y1197) Cell Signaling 4561 pHER3 (Y1289) Cell Signaling 4791 pGSK-3b (S9) Cell Signaling 9331 c-Met Cell Signaling 8198 c-Met Cell Signaling 3127 p-Met (Y1245/1235) Cell Signaling 3126 p-AKT (S473) Cell Signaling 4060 Ubiquitin (P4D1) Cell Signaling 3936 p230 Trans Golgi BD Biosciences 611281 EEA1 BD Biosciences 610456 Clathrin Heavy Chain SantaCruz sc-12734 Alexa Fluor-546 conjugated anti-mouse ThermoFisher A-21143 Alexa Fluor-488 conjugated anti-rabbit ThermoFisher A-11008 HRP linked Mouse IgG GE Life Sciences NA931 HRP Linked Rabbit IgG GE Life Sciences NA934 Chemicals, Peptides, and Recombinant Proteins Cycloheximide Sigma C104450 MLN4924 Sigma 5.05477 PR-619 LifeSensors SI9619 EZ-link Sulfo-NHS-SS-Biotin Thermo 21331 Pierce NeutrAvidin Agarose beads Thermo 29200 Agarose-TUBE 1 LifeSensors UM401 Protein A-Sepharose 4B, Fast Flow beads Sigma P9424 Recombinant Human HGF Protein R&D Systems 294-HG-250 Critical Commercial Assays CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) Promega G5421 Experimental Models: Cell Lines OVCAR8 Joyce Liu, Dana Farber MDA-MB-231 ATCC HTB-26 HeLa ATCC CCL-2 HCC827 ATCC CRL-2868 H3255 Katerina Politi, Yale H1975 ATCC CRL-5908 SKBr3 ATCC HTB-30 GTL16 F. Maina, Developmental Biology Institute of Marseille-Luminy Hs746T ATCC HTB-135 Oligonucleotides Clathrin Heavy Chain siRNA SantaCruz sc-35067 Software and Algorithms Image Lab 6.0 Biorad N/A Graphpad Prism N/A Open in a separate window Key Resources Table

Techniques: Ubiquitin Proteomics, Recombinant, Proliferation Assay, Software