Journal: Analytical chemistry

Article Title: Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels

doi: 10.1021/acs.analchem.9b04485

Figure Lengend Snippet: Quantification of EV concentrations with the 300-nm CuS-MG in 1× PBS or Serum. Calibration curves in 1×PBS using a) anti-CD63, b) anti-HER2; and in serum using c) anti-CD63 or d) anti-HER2. CL0 = chemiluminescence without EVs. [ABEI] = 0.5 mM, [H2O2] =1 mM, pH=11.

Article Snippet: The biotinylated mouse anti-human ErbB2/HER2 (Recombinant Monoclonal Human IgG 1 Clone Hu5), mouse anti-human CD63 (Clone mem-259), and biotinylated mouse anti-human CD63 (Clone NVG-2) was obtained from R&D systems, Sino Biological, and BioLegend, respectively.

Techniques:

Journal: Analytical chemistry

Article Title: Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels

doi: 10.1021/acs.analchem.9b04485

Figure Lengend Snippet: Detection of cell released EVs using CuS-MG. The cell culture medium was collected from three cell lines: MCF-10A, MDA-MB-231 and SK-BR-3. a) Chemiluminescence resulted from EV detection in the culture media of three cell lines targeting CD63 and anti-HER2; b) PCA plot using the chemiluminescence data shown in a); c) Linear correlation between CD63 quantification results obtained by ELISA and the chemiluminescent signals from CuS-MG in our assay targeting CD63. [ABEI] = 0.5 mM, [H2O2] = 1 mM, pH=11.

Article Snippet: The biotinylated mouse anti-human ErbB2/HER2 (Recombinant Monoclonal Human IgG 1 Clone Hu5), mouse anti-human CD63 (Clone mem-259), and biotinylated mouse anti-human CD63 (Clone NVG-2) was obtained from R&D systems, Sino Biological, and BioLegend, respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A Analysis of the immunohistochemical pathological reports for HER2 of 13 CRC patients in Jiangsu Provincial Hospital. B Western blot analysis of HER2 expression in murine and human CRC cell lines. C Expression and localization of HER2 detected by immunofluorescence on MC38 HER2 , MC38 Ctrl , CT26 HER2 , and CT26 Ctrl cells. D The mRNA expression of HER2 was measured by qPCR. ** P < 0.01. E The expressions of HER2 on the cell membrane were detected using flow cytometry. F The expression and localization of HER2 in HCT116 and HT29 cells were determined by immunofluorescence. Scale bars, 10 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Immunohistochemical staining, Western Blot, Expressing, Immunofluorescence, Flow Cytometry

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A In vitro growth inhibition assay for HCT116 cells treated with MMAE and Paclitaxel for 24 h. B In vitro growth inhibition assay for HCT116 and HT29 cells. C In vitro growth inhibition assay for MC38 HER2 and MC38 Ctrl cells treated with RC48 for 48 h. D In vitro growth inhibition assay for MC38 Ctrl and MC38 HER2 cells treated with MMAE for 24 h. E Flow cytometry analysis of cell cycle after treatment with different concentrations of RC48 for 48 h. F Clonogenic assay of HCT116, CT26 HER2 and MC38 HER2 treated with RC48. Data represent the mean ± SEM of three replicates. * P < 0.05, ** P < 0.01, versus as indicated. n.s. not significant.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: In Vitro, Growth Inhibition Assay, Flow Cytometry, Clonogenic Assay

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A MC38 Ctrl cells were treated with different concentrations of Disitamab (0, 10, 30, 100 μg/ml) for 48 h, and the expression of p-TBK1, p-IRF3, TBK1, IRF3 protein was detected by immunoblotting. B MC38 HER2 and MC38 Ctrl cells were treated with 100 μg/ml RC48 for 48 h and 30 μM CPT11 for 12 h and the expression of p-TBK1, p-IRF3, TBK1, IRF3 protein was detected by immunoblotting. C HCT116 was treated with 100 μg/ml RC48 for 48 h and 30 μM CPT11 for 12 h. D The expressions of HER2 on the cell membrane were detected using flow cytometry. MFI Mean fluorescence intensity. E Co-immunoprecipitation of endogenous HER2 and STING in HCT116. F The expression and localization of HER2 in MC38 HER2 were detected by immunofluorescence. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 10 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Expressing, Western Blot, Flow Cytometry, Fluorescence, Immunoprecipitation, Immunofluorescence

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A – E MC38 HER2 (1 × 10 6 ) cells were inoculated in the C57BL/6 mice. Drugs were given as described in Materials and Methods. B , C Tumor volume and body weight were measured every two days. D , E Solid tumors were separated and weighed after the mice were sacrificed. The data represent the mean ± SEM of 10 mice per group. F Paraffin sections of tumor tissues were analyzed by H&E staining, PCNA immunohistochemistry staining, and TUNEL fluorescent staining. G The survival of tumor-bearing mice. H – M Same experiment with CT26 HER2 in BALB/c mice. The data represent the mean ± SEM of 7 mice per group. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 50 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Staining, Immunohistochemistry, TUNEL Assay

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A Immunofluorescence staining for p-TBK1 and p-IRF3 in tumor tissue in C57BL/6. B , C Immunohistochemical detection of IFN-β secretion in tumor tissue in BALB/c and C57BL/6. D LDH release assay was performed using Tils achieved from the syngeneic model of CT26 HER2 as described in material and methods. E , F Examination of CD3 + , CD8 + , IFN-γ secretion in the tumor by flow cytometry in BALB/c and C57BL/6. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 100 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Lactate Dehydrogenase Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: RC48 exhibits the capability to bind to HER2 receptors and undergo internalization, subsequently releasing the toxin MMAE. MMAE inhibits tubulin, leading to the disruption of the cell cycle and resulting in the efficient elimination of tumor cells. Moreover, through its interaction with HER2, RC48 can alleviate the inhibition induced by internalized HER2 on the STING pathway and its downstream signaling. These combined mechanisms enable RC48 to effectively impede the proliferation of HER2-positive tumors while also enhancing their sensitivity to immunotherapy.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Inhibition

Journal: iScience

Article Title: From byproduct to biotherapeutic: Comparative study of buffy coats and leukoreduction system chambers for NK cell-based immunotherapies

doi: 10.1016/j.isci.2026.114907

Figure Lengend Snippet: Functional characterization of NK cells (A) Degranulation capacity of NK cells following a 2 h co-culture with K562 leukemia cells, measured by surface CD107a expression ( n = 5). (B and C) Europium-based cytotoxicity assay assessing the specific lysis of K562 target cells at E:T ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 on day 7 (B) and day 14 (C) of culture to monitor functional decline over time ( n = 6 for both time points). Here, all comparisons were not significant and are summarized as such (n.s.: p > 0.05). (D) IFN-γ release following 16 h of co-culture of NK cells with K562 cells, quantified via Luminex assay ( n = 5). (E) Kinetic live-cell killing assay using the Incucyte monitoring of Nuclight Red signal loss in K562 cells over 72 h at an E:T ratio of 1:1 ( n = 6). (F) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis thresholds during Incucyte co-culture with K562 cells. (G) CD107a degranulation assay of NK cells with or without pre-incubation with trastuzumab (anti-HER2 antibody) during a 2 h co-culture with MDA-MB-453 cells ( n = 5). (H) Incucyte-based cytotoxicity assay shows the real-time lysis of MDA-MB-453 cells by trastuzumab-loaded NK cells at an E:T ratio of 1:1 over 72 h ( n = 6). (I) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the ADCC assay. (n varies as not all samples reached the benchmark values within the observation period). All ADCC experiments have been performed after 14 days of in vitro NK cell expansion. N/A indicates that statistical analysis was not possible for this group. 9J) CD107a degranulation of unmodified NK cells and ErbB2-CAR-NK cells after 2 h co-culture with MDA-MB-453 cells ( n = 5). (K) Incucyte-based cytotoxicity assay showing killing dynamics of ErbB2-CAR-NK cells against MDA-MB-453 cells at an E:T ratio of 1:1 over 72 h ( n = 6). (L) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the CAR-mediated cytotoxicity assay. (n varies as not all samples reached the benchmark values within the observation period). All CAR-killing experiments have been performed after 14 days of in vitro NK cell expansion. Unless otherwise stated, n refers to biologically independent donor samples and is indicated per group. Data are shown as mean ± SEM. p-values were determined by Student’s t test with Welsh correction (A, D, F, G, I, J, and L) or by one-way ANOVA (B and C) with Tukey post-test (K and L).

Article Snippet: Mouse Monoclonal anti-CD340 (Her2) , Miltenyi Biotec , Cat# 130-124-474.

Techniques: Functional Assay, Co-Culture Assay, Expressing, Cytotoxicity Assay, Lysis, Luminex, Degranulation Assay, Incubation, ADCC Assay, In Vitro

Journal: Scientific Reports

Article Title: HLA-DR cancer cells expression correlates with T cell infiltration and is enriched in lung adenocarcinoma with indolent behavior

doi: 10.1038/s41598-021-93807-3

Figure Lengend Snippet: Mass cytometry antibody panel for lung adenocarcinoma.

Article Snippet: HER2 , 148Nd , Surface , 29D8 , Fluidigm , 3148011A.

Techniques: Mass Cytometry

Journal: Journal of Nuclear Medicine

Article Title: Advancing Targeted Radionuclide Therapy Through the National Cancer Institute’s Small Business Innovation Research Pathway

doi: 10.2967/jnumed.118.214684

Figure Lengend Snippet: NCI SBIR–Funded TRT Contracts for 2015

Article Snippet: 2016 , Rockland Immunochemicals, Inc. , Phase I , HER2-positive cancers , 68 Ga and 177 Lu , HER2 and digoxigenin , Phase I: Develop bispecific recombinant single-domain antibodies with one arm targeting cancer biomarker HER2 and another arm against digoxigenin; investigate in vivo clearance, tumor accumulation, stability, and ability to capture digoxigenylated fluorescence dyes and radionuclides.

Techniques: In Vitro, In Vivo, Marker, Fluorescence, Drug discovery, Imaging

Journal: Journal of Nuclear Medicine

Article Title: Advancing Targeted Radionuclide Therapy Through the National Cancer Institute’s Small Business Innovation Research Pathway

doi: 10.2967/jnumed.118.214684

Figure Lengend Snippet: NCI SBIR–Funded TRT Contracts from 2016 to 2017

Article Snippet: 2016 , Rockland Immunochemicals, Inc. , Phase I , HER2-positive cancers , 68 Ga and 177 Lu , HER2 and digoxigenin , Phase I: Develop bispecific recombinant single-domain antibodies with one arm targeting cancer biomarker HER2 and another arm against digoxigenin; investigate in vivo clearance, tumor accumulation, stability, and ability to capture digoxigenylated fluorescence dyes and radionuclides.

Techniques: Radioactivity, Drug discovery, Recombinant, Biomarker Discovery, In Vivo, Fluorescence, Binding Assay, Labeling, In Vitro, Produced, Software

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Counting of SK-BR3 cells, stained with DAPI (cell nuclei) and immunolabeled with SiO 2 @(QD635-Dex)-(anti-HER2 TAC). (A) SK-BR3 cells only: (i–v) frames from smartphone FC videos of different concentrations of suspended cells. For the most dilute suspension, the arrow indicates a single cell. An intensity profile across selected cells can be found in Figure S13 . The image in (i) is a zoomed section of (v) with one (of several) SK-BR3 cells circled. Correlation plots (vi) of HER2-positive SK-BR3 cell counts derived from the smartphone FC versus a commercial cell counter. The diagonal line has a slope of unity. (B) Target SK-BR3 cells and background MDA-MB-231 cells: (i–v) frames from smartphone FC videos of mixed suspensions of SK-BR3 cells (variable amount) and MDA-MB-231 cells (approximately constant amount; DAPI stained). An intensity profile across selected cells of each type can be found in Figure S13 . Video S3 is a 10 s clip from one of the smartphone FC videos. The image in (i) is a zoomed section of (v) with one example each of a SK-BR3 cell and a MDA-MB-231 cell circled. Correlation plots of cell counts (vi) measured with a smartphone FC versus a commercial cell counter. Frame image brightness has been digitally increased for display purposes.

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques: Staining, Immunolabeling, Suspension, Derivative Assay

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Assessment of multiple colors of QDs. (A) PL emission spectra of QD490, QD540, QD585, QD605, QD635, and QD650 (solid lines). The approximate RGB color filter transmission spectra for the smartphone are overlaid (dashed lines; data courtesy of Olivier Burggraaff ). (B) Frames from smartphone FC videos showing SK-BR3 cells immunolabeled with SiO 2 @(QDλ-CM-Dex)-(anti-HER2) for QD540, QD585, QD605, or QD650. Frame image brightness has been digitally increased for display purposes. Intensity profiles across selected cells of each color of QD can be found in Figure S16 . (C) Plots of the G/B and R/B intensity ratios for individual SK-BR3 cells labeled with these four colors of SiO 2 @(QDλ-CM-Dex)-(anti-HER2). (D) Plots of the R, G, and B channel intensity values for individual SK-BR3 cells nonspecifically labeled with GSH-QD490, GSH-QD540, or GSH-QD635 (none as a SiO 2 @QD assembly).

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques: Transmission Assay, Immunolabeling, Labeling

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Counting of SK-BR3 and MDA-MB-231 cells in parallel using SiO 2 @(QD540-CM-Dex)-(anti-HER2) and SiO 2 @(QD650-CM-Dex)-(anti-MUC1), respectively. (A) Cartoon schematic of the experiment (not drawn to scale). (B) Example of a frame from a smartphone FC video. Frame image brightness has been digitally increased for display purposes. An intensity profile across selected cells of each type can be found in Figure S17 . Video S4 is a 20 s clip from one of the smartphone FC videos. (C) Examples of normalized G/B and R/B intensity ratios and cell classifications for five samples with an increasing number of MDA-MB-231 cells and an approximately constant number of SK-BR3 cells (see Figure S18 for additional data sets). Normalization of intensity ratios assisted with application of the SVM training set to the experimental data (see the SI for details). (D) Correlation plots for the measured and expected numbers of SK-BR3 and MDA-MB-231 cells, and for the measured and expected ratios of MDA-MB-231 and SK-BR3 cells. This plot includes the samples in panel (C) and in Figure S18 .

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques:

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Identification of selected breast cancer cell types (SK-BR3, MDA-MB-231, and MCF-7) using SiO 2 @(QD540-CM-Dex)-(anti-HER2), SiO 2 @(QD585-CM-Dex)-(anti-MUC1), and SiO 2 @(QD650-CM-Dex)-(anti-ER). (A) Cartoon schematic of the experiment (not drawn to scale). (B) Examples of G/B and R/B intensity ratios for a control sample without cells and suspensions of each cell type. The shaded region in the plots corresponds to the mean of the control sample data points plus four standard deviations along each intensity ratio axis. The circled regions highlight the dominant vertical, diagonal, and horizontal spread of cell counts for SK-BR3, MDA-MB-231, and MCF-7 cells, respectively.

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques: Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Inhibition of Tumor Growth and Immunomodulatory Effects of Flavonoids and Scutebarbatines of Scutellaria barbata D. Don in Lewis-Bearing C57BL/6 Mice

doi: 10.1155/2015/630760

Figure Lengend Snippet: Effect of flavonoids and scutebarbatines in SB on the levels of immune factors in Lewis tumor-bearing mice. All data were expressed as means ± SD ( n = 9).

Article Snippet: Antibodies to these immunoregulatory factors including IL-10, IL-17, FOXP3, IFN- γ , IL-2, ROR γ t, TGF- β 1, IL-6, and β -actin were obtained from Wuhan BOSTER Biological Engineering Co., Ltd. (Wuhan, China).

Techniques:

Journal: International Journal of Oncology

Article Title: Although c-MYC contributes to tamoxifen resistance, it improves cisplatin sensitivity in ER-positive breast cancer

doi: 10.3892/ijo.2020.4987

Figure Lengend Snippet: Establishment of TAM-resistant breast cancer cells. (A) TAM-resistant cells exhibited a more elongated, spindle-shaped morphology compared with the respective parental cells. Magnification, ×200. (B) Survival of TAM-resistant cells and the respective parental cells was evaluated using a Cell Counting kit-8 assay following treatment with various concentrations of TAM for 24 h. (C) Western blot analysis (D) and densitometry analysis of ERα and HER2 expression in MCF-7, MCF-7R, T47D and T47DR cells. Data are presented as the means ± standard deviation of the mean of 3 repeats. * P<0.05, ** P<0.01, ***P<0.001 vs. respective parental cells. TAM, tamoxifen.

Article Snippet: Non-specific binding sites were blocked by incubating the membranes with 5% non-fat milk, after which the membranes were incubated overnight at 4°C with the primary antibodies: ERα (1:1,000); HER2 (1:1,000); AKT (1:1,000); p-AKT (1:1,000); c-MYC (1:1,000); cyclin D1 (1:1,000); vimentin (1:1,000); E-cadherin (1:1,000); p21 (1:500); β-catenin (1:500); GAPDH (1:1,000) Membranes were subsequently incubated with a horseradish peroxidase-conjugated anti-rabbit/mouse immunoglobulin G secondary antibody (1:1,000; cat. no. BA1075 and BA1051; Wuhan Boster Biological Technology, Ltd.).

Techniques: Cell Counting, Western Blot, Expressing, Standard Deviation

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: