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: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Plasma levels of Foxp3. Peripheral blood mononuclear cells were collected from each group, and the proportions of CD4 + CD25 + Foxp3 + T reg /CD4 + T-cells were analyzed by flow cytometry, and quantified using FlowJo 7.6.1 software. (A) Histogram. Data are presented as the mean ± standard deviation. *P<0.01, vs. the AS group. (B) Negative group, (C) AS group, (D) atorvastatin group and (E) IL-35 group. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35; APC, allophycocyanin; PE, phycoerythrin.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Clinical Proteomics, Flow Cytometry, Software, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: IL-35 improves T reg -mediated immune suppression in atherosclerotic mice

doi: 10.3892/etm.2016.3649

Figure Lengend Snippet: Levels of Foxp3 in atherosclerotic lesions. The deposition of Foxp3 in arteries from various groups was detected by immunohistochemistry (magnification, ×400). Positive Foxp3 was shown as brown nuclei. In the (A) negative and (B) AS groups, the deposition of Foxp3 in the lesions was minimal. Conversely, Foxp3 deposition was increased in the lesions of the (C) atorvastatin and (D) IL-35 groups, as compared with the AS group. (E) This was shown to be significant following quantification. There was no significant difference in the levels of Foxp3 between the atorvastatin and IL-35 groups. *P<0.01, vs. the negative control. Foxp3, forkhead box protein 3; AS, atherosclerosis; IL-35, interleukin-35.

Article Snippet: Anti-Foxp3 antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Immunohistochemistry, Negative Control

Journal: Oncology Research

Article Title: High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl) phenyl]-1H-isoindole-1,3(2H)-dione (C3), as a novel EGFR—HER2 dual inhibitor in gastric tumors

doi: 10.32604/or.2023.043139

Figure Lengend Snippet: Protein-ligand docking of known kinase inhibitors. (a) Predicted binding pose of AQ4 (erlotinib) to EGFR (green), in comparison with experimental binding pose from 4hjo (red). (b) Binding pose prediction based on protein-ligand docking of 03P with HER2 kinase (3rcd, green), in comparison with experimentally derived binding pose (red).

Article Snippet: EGFR (T790M/L858R) Kinase Assay Kit (Catalog # 40322) and HER2 Kinase Assay Kit (Catalog # 40721) were purchased from BPS Bioscience, SD, USA.

Techniques: Binding Assay, Comparison, Derivative Assay

Journal: Oncology Research

Article Title: High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl) phenyl]-1H-isoindole-1,3(2H)-dione (C3), as a novel EGFR—HER2 dual inhibitor in gastric tumors

doi: 10.32604/or.2023.043139

Figure Lengend Snippet: High-throughput virtual screening identify targeted compounds for EGFR and HER2. (a) Predicted binding scores for the ChemBridge compounds against EGFR kinase. Histogram includes compounds having docking score >2 standard deviation from the mean docking scores (−9.7 kcal/mol). (b) Docking scores, >2 standard deviation from the mean, for top EGFR compounds from (a) screened against HER2 kinase. (c) Predicted docking score for compound C3 and a known inhibitor AQ4 against EGFR. (d) Predicted docking score for compound C3 and a known inhibitor 03P against HER2. (e) Interacting residues between compound C3 and EGFR. (f) 2D representation of compound C3 interactions at the kinase site of EGFR. (g) Interacting residues between compound C3 and HER2. (h) 2D representation of compound C3 interactions at the kinase site of HER2.

Article Snippet: EGFR (T790M/L858R) Kinase Assay Kit (Catalog # 40322) and HER2 Kinase Assay Kit (Catalog # 40721) were purchased from BPS Bioscience, SD, USA.

Techniques: High Throughput Screening Assay, Binding Assay, Standard Deviation

Journal: Oncology Research

Article Title: High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl) phenyl]-1H-isoindole-1,3(2H)-dione (C3), as a novel EGFR—HER2 dual inhibitor in gastric tumors

doi: 10.32604/or.2023.043139

Figure Lengend Snippet: Molecular dynamics simulation of the HER2-C3 complex (a) Snapshot of trajectories taken at different time points during the simulation. (b) Predicted ligand root mean square deviation of compound C3 when bound to HER2. (c) Predicted average hydrogen bonds between compound C3 and HER2 for a 100 ns simulation.

Article Snippet: EGFR (T790M/L858R) Kinase Assay Kit (Catalog # 40322) and HER2 Kinase Assay Kit (Catalog # 40721) were purchased from BPS Bioscience, SD, USA.

Techniques:

Journal: Oncology Research

Article Title: High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl) phenyl]-1H-isoindole-1,3(2H)-dione (C3), as a novel EGFR—HER2 dual inhibitor in gastric tumors

doi: 10.32604/or.2023.043139

Figure Lengend Snippet: MM/PBSA-based binding free energy predictions for compound C3 (a) Predicted binding free energy (ΔG binding ) for compound C3 to EGFR and HER2 calculated based on MM/PBSA scoring from simulation trajectories (total cutoff value: −63.59 kJ/mol for EGFR and −87.42 kJ/mol for HER2). (b) Residue level binding energy contribution for the EGFR-C3 complex (cut off value −6 kJ/mol). (c) Residue level binding energy contribution for the HER2-C3 complex (cutoff value −6 kJ/mol). (d) MD simulation and MM/PBSA-based predicted binding free energy comparison between compound C3 and dacomitinib bound to EGFR (e) MD simulation and MM/PBSA-based predicted binding free energy comparison between compound C3 and dacomitinib bound to HER2.

Article Snippet: EGFR (T790M/L858R) Kinase Assay Kit (Catalog # 40322) and HER2 Kinase Assay Kit (Catalog # 40721) were purchased from BPS Bioscience, SD, USA.

Techniques: Binding Assay, Residue, Comparison

Journal: Oncology Research

Article Title: High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl) phenyl]-1H-isoindole-1,3(2H)-dione (C3), as a novel EGFR—HER2 dual inhibitor in gastric tumors

doi: 10.32604/or.2023.043139

Figure Lengend Snippet: IC 50 of C3 against (a) EGFR and (b) HER2 enzymes. GI 50 of C3 in controlling proliferation of (c) KATO III and (d) SNU-5 cells.

Article Snippet: EGFR (T790M/L858R) Kinase Assay Kit (Catalog # 40322) and HER2 Kinase Assay Kit (Catalog # 40721) were purchased from BPS Bioscience, SD, USA.

Techniques:

Journal: Cell Reports Medicine

Article Title: Synergically enhanced anti-tumor immunity of in vivo panCAR by circRNA vaccine boosting

doi: 10.1016/j.xcrm.2025.102250

Figure Lengend Snippet: CircRNA CAR efficiently expressed CAR proteins and mediated remarkable tumor killing (A) Schematic representation of circRNA Anti-HER2-CAR circularization via group I intron autocatalysis. (B and C) Detecting the expression of CAR proteins in HEK293T cells after circRNA Anti-HER2-CAR transfection via western blot (B) and flow cytometry (C). (D) Comparative analysis of CAR expression levels from circRNA Anti-HER2-CAR , 1mΨ-mRNA Anti-HER2-CAR , and unmodified mRNA Anti-HER2-CAR in HEK293T cells. (E–G) Optimization of circRNA Anti-HER2-CAR encoding CAR in Jurkat (E), THP-1 (F), and J774A.1 (G). (H) Detection of CAR expression in primary T cells using flow cytometry. (I–K) Cytotoxic effects of primary T cells transfected with circRNA Anti-HER2-CAR on SK-OV-3 (I), B16F10-HER2 (J), and 4T1-HER2 (K) tumor cells. (L–N) Cytotoxic effects of Jurkat cells transfected with circRNA Anti-HER2-CAR on SK-OV-3 (L), B16F10-HER2 (M), and 4T1-HER2 (N) tumor cells. (O–Q) Cytotoxic effects of THP-1 cells transfected with circRNA Anti-HER2-CAR on SK-OV-3 (O), B16F10-HER2 (P), and 4T1-HER2 (Q) tumor cells. In (C)–(Q), data were presented as mean ± SEM ( n = 3). An unpaired two-sided Student’s t test was performed for comparison; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. See also ; .

Article Snippet: The 96-well plates (Corning, #9018) were coated with recombinant human HER2 protein (MCE, #HY- P70254 ) diluted in coating solution (Bioss, #C04-01001) at 4°C overnight.

Techniques: Expressing, Transfection, Western Blot, Flow Cytometry, Comparison

Journal: Cell Reports Medicine

Article Title: Synergically enhanced anti-tumor immunity of in vivo panCAR by circRNA vaccine boosting

doi: 10.1016/j.xcrm.2025.102250

Figure Lengend Snippet: Macrophages exhibited efficient tumor phagocytosis and pro-inflammatory polarization induced by circRNA CAR (A and B) Phagocytosis of THP-1 cells transfected with circRNA Anti-HER2-CAR against SK-OV-3 (A) and MC38-HER2 (B) cells. (C) Phagocytosis of J774A.1 cells transfected with circRNA Anti-HER2-CAR against CT26-HER2 cells. (D and E) Effects of circRNA Anti-HER2-CAR on iNOS (D) and CD206 (E) expression in J774A.1. (F and G) Effects of circRNA Anti-HER2-CAR on iNOS (F) and CD206 (G) expression in THP-1 cells. (H) Volcano plot illustrating differentially expressed genes in THP-1 cells. (I) Heatmap depicting gene expression patterns in THP-1 cells ( n = 2). (J) Bubble chart of relevant biological processes via Gene Ontology (GO) analysis ( n = 2). Bubble size represents the number of genes. In (A)–(G), data were presented as mean ± SEM ( n = 3). An unpaired two-sided Student’s t test was conducted for comparison; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. See also .

Article Snippet: The 96-well plates (Corning, #9018) were coated with recombinant human HER2 protein (MCE, #HY- P70254 ) diluted in coating solution (Bioss, #C04-01001) at 4°C overnight.

Techniques: Transfection, Expressing, Gene Expression, Comparison

Journal: Cell Reports Medicine

Article Title: Synergically enhanced anti-tumor immunity of in vivo panCAR by circRNA vaccine boosting

doi: 10.1016/j.xcrm.2025.102250

Figure Lengend Snippet: Screening for immunocyte-tropic LNPs that efficiently delivered circRNAs into immune cells in mice (A) Schematic representation of the LNP-circRNA complex. (B and C) The size distribution (B) and zeta potential (C) of the LNP-circRNA Luciferase complex. (D and E) Bioluminescence imaging in vivo (D) or ex vivo (E) of BALB/c mice intravenously injected with PBS or LNP-circRNA Luciferase . (F and G) Bioluminescence imaging in vivo (F) or ex vivo (G) of BALB/c mice intravenously injected with PBS or SORT-circRNA Luciferase . (H) Evaluation of targeting efficiency of SORT-circRNA Cre in the spleen of reporter mice. (I) Detection of anti-HER2-CAR expression at different time points in various immune cells of mouse spleen. In (H) and (I), data were presented as mean ± SEM, an unpaired two-sided Student’s t test was performed for comparison; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. See also .

Article Snippet: The 96-well plates (Corning, #9018) were coated with recombinant human HER2 protein (MCE, #HY- P70254 ) diluted in coating solution (Bioss, #C04-01001) at 4°C overnight.

Techniques: Zeta Potential Analyzer, Luciferase, Imaging, In Vivo, Ex Vivo, Injection, Expressing, Comparison

Journal: Cell Reports Medicine

Article Title: Synergically enhanced anti-tumor immunity of in vivo panCAR by circRNA vaccine boosting

doi: 10.1016/j.xcrm.2025.102250

Figure Lengend Snippet: CircRNA CAR efficiently inhibited tumor growth and improved survival time in mice (A) Schematic illustration of intravenous PBS, LNP-circRNA Ctrl , or SORT-circRNA Anti-HER2-CAR treatment in the CT26-HER2 tumor model. (B) Tumor growth curves for CT26-HER2 tumor-bearing mice treated as indicated in (A). (C) Schematic illustration of intratumoral PBS, LNP-circRNA Ctrl , or LNP-circRNA Anti-HER2-CAR treatment in the CT26-HER2 tumor model. (D and E) Tumor growth curves (D) and survival curves (E) of CT26-HER2 tumor-bearing mice treated as indicated in (C). (F) In vivo bioluminescence imaging of CT26-HER2 tumor-bearing mice treated as indicated in (C). (G) The quantified signal intensity of bioluminescence imaging in (F). (H) Schematic illustration of intratumoral PBS, LNP-circRNA Ctrl , or LNP-circRNA Anti-HER2-CAR treatment in the 4T1-HER2 tumor model. (I) Tumor growth curves of 4T1-HER2 tumor-bearing mice treated as indicated in (H). (J) Tumor growth curves of individual 4T1-HER2 tumor-bearing mice treated as indicated in (H). (K) Schematic illustration of intratumoral PBS, LNP-circRNA Ctrl , or LNP-circRNA CAR treatment in the MC38-HER2 tumor model. (L and M) Tumor growth curves (L) and survival curves (M) of MC38-HER2 tumor-bearing mice treated as indicated in (K). In (B), (D), (I), and (L), data were represented as the mean ± SEM, the tumor growth curves were calculated by two-way ANOVA analysis ( n = 6). In (E) and (M), data were represented as the mean ± SEM, the survival curves were calculated by Kaplan-Meier simple survival analysis ( n = 6). In (G), data were represented as the mean ± SEM, an unpaired two-sided Student’s t test was conducted for comparison. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Article Snippet: The 96-well plates (Corning, #9018) were coated with recombinant human HER2 protein (MCE, #HY- P70254 ) diluted in coating solution (Bioss, #C04-01001) at 4°C overnight.

Techniques: In Vivo, Imaging, Comparison

Journal: Cell Reports Medicine

Article Title: Synergically enhanced anti-tumor immunity of in vivo panCAR by circRNA vaccine boosting

doi: 10.1016/j.xcrm.2025.102250

Figure Lengend Snippet: In vivo panCAR reshaped the tumor microenvironment to a pro-inflammatory state (A) Changes in the proportion of immune cells of spleen via flow cytometry. CD8 + T cells, CD8 + Tem cells, CD8 + Tcm cells, or CD4 + T cells were gated from CD45 + cell population, and Treg cells were gated from CD4 + T cell population. (B) Flow cytometric analysis of changes in the proportion of infiltrating immune cells in tumors. CD8 + T cells, CD4 + T cells, or MHC II + macrophages were gated from CD45 + cell population, and Treg cells were gated from CD4 + T cell population. (C and D) H&E staining (C) or IHC staining (D) of tumor tissue sections obtained from CT26-HER2 tumor-bearing mice after PBS, SORT-LNP-circRNA Ctrl , or SORT-LNP-circRNA Anti-HER2-CAR treatment. The integrated density of IHC staining was quantified using ImageJ. (E) Heatmap of gene expression patterns of immune cells extracted from tumor tissues ( n = 2). (F) Bubble chart of relevant biological processes through GO analysis ( n = 2). The size of the bubbles represented the number of genes. (G) Gene set enrichment analysis (GSEA) showing enriched pathways in immune cells extracted from tumor tissues ( n = 2). In (A), (B), and (D), data were represented as the mean ± SEM; an unpaired two-sided Student’s t test was conducted for comparison; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. Each symbol represents an individual mouse. See also and .

Article Snippet: The 96-well plates (Corning, #9018) were coated with recombinant human HER2 protein (MCE, #HY- P70254 ) diluted in coating solution (Bioss, #C04-01001) at 4°C overnight.

Techniques: In Vivo, Flow Cytometry, Staining, Immunohistochemistry, Gene Expression, Comparison

Journal: Cell Reports Medicine

Article Title: Synergically enhanced anti-tumor immunity of in vivo panCAR by circRNA vaccine boosting

doi: 10.1016/j.xcrm.2025.102250

Figure Lengend Snippet: CircRNA vaccine synergistically boosted the anti-tumor activity of in vivo panCAR (A) Schematic illustration of circRNA VAC design. EPM, the endocytosis prevention motif; EABR, the ESCRT- and ALIX-binding region domain. (B) Flow cytometric analysis detecting the translation of circRNA HER2 in HEK293T cells ( n = 3). (C) Electron microscopy showing the vesicles secreted by HEK293T cells transfected with circRNA HER2-EPM-EABR . (D) Measurement of the endpoint titer of HER2-specific IgG with ELISA. (E) Schematic illustration of 4T1-HER2 tumor-bearing mice receiving PBS (intravenously), circRNA CAR (intravenously), circRNA VAC (intramuscularly), or circRNA CAR (intravenously) plus circRNA VAC (intramuscularly) combined therapy ( n = 5). (F) Tumor growth curves of overall mice treated as indicated in (E). (G) Tumor growth curves of individual mouse treated as indicated in (E). (H) Schematic illustration of B16F10-HER2 tumor-bearing mice receiving PBS (intravenously), circRNA CAR (intravenously), circRNA VAC (intramuscularly), or circRNA CAR (intravenously) plus circRNA VAC (intramuscularly) combined therapy ( n = 5). (I) Tumor growth curves of overall mice treated as indicated in (H). (J) Tumor growth curves of individual mouse treated as indicated in (H). (K) Survival curves of B16F10-HER2 tumor-bearing mice treated as indicated in (H). In (B), data were represented as the mean ± SEM; an unpaired two-sided Student’s t test was performed for comparison. In (D), data were represented as the geometric mean ± geometric SD; an unpaired two-sided Student’s t test was performed for comparison. In (F) and (I), data were represented as the mean ± SEM, and the tumor growth curves were calculated by two-way ANOVA analysis. In (K), the survival curves were calculated by Kaplan-Meier simple survival analysis. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. See also ; .

Article Snippet: The 96-well plates (Corning, #9018) were coated with recombinant human HER2 protein (MCE, #HY- P70254 ) diluted in coating solution (Bioss, #C04-01001) at 4°C overnight.

Techniques: Activity Assay, In Vivo, Binding Assay, Electron Microscopy, Transfection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cell Reports Medicine

Article Title: Synergically enhanced anti-tumor immunity of in vivo panCAR by circRNA vaccine boosting

doi: 10.1016/j.xcrm.2025.102250

Figure Lengend Snippet: In vivo panCAR enhanced the anti-tumor activity via antibody-mediated cellular cytotoxicity (A) Measurement of HER2-specific IgG, IgG1, IgG2A, IgG2B, or IgG2C-binding antibodies with ELISA ( n = 5 or 6). (B and C) HER2-specific antibodies in (A) mediated cellular cytotoxicity against SK-OV-3 (B) and MC38-HER2 (C) tumor cells in J774A.1. (D and E) HER2-specific antibodies in (A) mediated cellular cytotoxicity against SK-OV-3 (D) and MC38-HER2 (E) tumor cells in RAW 264.7. (F) Tumor growth curves of overall CT26 tumor-bearing mice treated with PBS, in vivo panCAR-VAC, or in vivo panCAR-VAC plus anti-NK1.1 antibodies to deplete NK cells ( n = 5). (G) Tumor growth curves of individual mouse treated as indicated in (F). (H) Survival curves of mice treated as indicated in (F). (I) Tumor growth curves of overall CT26 tumor-bearing mice treated with PBS, in vivo panCAR-VAC, or in vivo panCAR-VAC plus anti-CSF1R antibody to deplete macrophages ( n = 5). (J) Tumor growth curves of individual mouse treated as indicated in (I). (K) Survival curves of mice treated as indicated in (I). (L) The potential mechanism diagram of synergistic in vivo panCAR-VAC immunotherapy. In (A), data are shown as the mean ± SEM; In (B)–(E), data were represented as the mean ± SEM; an unpaired two-sided Student’s t test was performed for comparison. In (F) and (I), tumor growth curves were calculated by two-way ANOVA analysis ( n = 5). In (H) and (K), survival curves were calculated by Kaplan-Meier simple survival analysis ( n = 5). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. See also .

Article Snippet: The 96-well plates (Corning, #9018) were coated with recombinant human HER2 protein (MCE, #HY- P70254 ) diluted in coating solution (Bioss, #C04-01001) at 4°C overnight.

Techniques: In Vivo, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison

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: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 3 TWEAK regulated the Th17/Treg cell ratio in AC mice. (A) The effect of TWEAK knockdown on Th17 and Treg cell ratio in spleen of AC mice was observed by flow cytometry. (B-C) WB and IHC were employed to evaluate the expression levels of FoxP3 and RORγt in mice conjunctival tissue. (D) The effect of TWEAK knockdown on the levels of Th17 and Treg cytokines in mice spleen were evaluated by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shNC; ns, no significant difference

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Fig. 6 Inhibition of Nrf2/HO-1 signaling pathway affected Th17/Treg cell ratio in AC mice with TWEAK knockdown. (A) The effect of Nrf2 inhibitor on Th17/Treg cell ratio in AC mice with TWEAK knockdown was assessed by flow cytometry. (B-C) WB and IHC assays were employed to evaluate the expres sion of FoxP3 and RORγt in conjunctival tissue of AC mice. (D) The levels of Th17 and Treg cytokines in mice spleen were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. AC + AAV-shNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AC + AAV-shTWEAK

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Inhibition, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay