Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Expression, cytotoxic activity, and cytokine secretion of T cells expressing altered version of FcγRI. A, Illustration of the modified FcγRI structure with fused signaling domains: A2G (left), FcγRI composed of α-chain and homodimer of FcRγ chain; AG2G (middle), FcγRI α-chain with fused domain of FcRγ chain and homodimer of FcRγ; AGO2GO (right), FcγRI α-chain with fused FcRγ and OX40 domains and homodimer of FcRγ with fused OX40 domain. B, Flow cytometry analysis of A2G, AG2G, and AGO2GO expression following retroviral transduction of healthy donor T cells. C, Mean numbers of HT29 target cells expressing HER2 calculated by incuCyte software through 48-hour incubation with the three FcγRI-based receptor expressing T cells, with 1:1 E:T ratios with or without 6 μg/mL trastuzumab (data shows one of two experiments repeats, n = 4). Each sample is normalized to its cell number at time zero. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. D, Representative incuCyte images showing HER2-expressing HT29 cells, imaged and counted in live imaging system after 48 hours of incubation with different construct-expressing T cells with 1:1 E:T ratio. Scale bar is 100 μm. E, Human Luminex Discovery Assay measurement of cytokine levels in supernatants from 48 hours of culture of T cells 4:1 E:T ratio with HT29 cells expressing HER2 ( n = 4). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Activity Assay, Modification, Flow Cytometry, Retroviral, Transduction, Software, Incubation, Imaging, Construct, Luminex

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Characterization of AG2G-expressing T cells, activation, and cytotoxicity. A, ELISA measurement of cytokine levels of AG2G-expressing T cells after 48-hour incubation with HT29 cells expressing HER2. B, IFNγ and TNFα levels measured by ELISA in supernatants from 48-hour incubation of AG2G-expressing T cells and HT29 cells expressing HER2 in different E:T ratios. C, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with 0, 2.5, 5, 10 mg/mL IVIG, with or without 30 μg/mL trastuzumab ( n = 4). D, ELISA measurement of IFNγ levels of AG2G-expressing cells cocultured with HT29 cells expressing HER2 described in B . E, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with different concentrations of trastuzumab over 96 hours ( n = 2). F, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with different E:T ratios of AG2G-expressing T cells that were isolated for their CD4 (left) or CD8 (middle) population or with no isolation (right). G, ELISA measurement of cytokine levels from supernatants of 48-hour culture of 4:1 E:T ratios of isolated CD4, isolated CD8, or AG2G-expressing T cells with HT29 cells expressing HER2 and 30 μg/mL trastuzumab. Graphs show mean ±SD. Statistical significance was calculated using Student t test for a, two-way ANOVA with Sidak correction for multiple comparisons for B and C , two-way ANOVA with Tukey's correction for multiple comparisons for E and F . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Software, Isolation

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: AG2G-expressing T cells differentiate between cells expressing high and low antigen levels, are more specific, and less exhausted, compared with classic CAR T cells. A, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with transduced T cells in combination with antibodies (60 μg/mL each, n = 4). B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-transduced T cells incubated with HER2-expressing HT29 target cells, and with tumor-binding trastuzumab, or irrelevant antibodies cetuximab and rituximab (60 μg/mL). C, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. D, Normalized confluence of kidney epithelial cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell confluence at time zero. E, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D ( n = 4). F, ELISA measurement of IFNγ, granzyme B and TNFα levels in 24-hour supernatants of 10 5 AG2G-expressing T cells or CAR T cells incubated on immobilized trastuzumab or rHER2, in rising concentrations (mmol/mL) respectively. G, Confocal microscope imaging of internalization kinetics of PE-labeled IgG by AG2G-expressing cells (top), or PE-labeled HER2 by CAR T cells (bottom). H, PD-1, LAG-3, and TIM3 flow cytometry analysis of AG2G-expressing cells or CAR T cells after 48-hour incubation with or without 12 mmol/mL immobilized trastuzumab or HER2, respectively. Graphs show mean ± SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A , C , D , and H . Two-way ANOVA with Sidak correction for multiple comparisons for E and F . One-way ANOVA with Dunnett correction for multiple comparisons for B . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Software, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay, Microscopy, Imaging, Labeling, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Human T cells expressing AG2G exert specific tumor cytotoxicity while sparing normal cells. A, Flow cytometry expression analysis of HER2 on different tumor cell lines and primary human normal cells. B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-expressing T cells with different tumor cells or primary normal cells in 4:1 E:T ratio and 60 μg/mL trastuzumab (results shown are from 3 different donors combined, n = 12). C, Quantification of HER2 receptor numbers using DAKO QIFIKIT (Agilent) beads by flow cytometer. Individual populations of the calibration are gated, a linear regression is calculated from the MFI and the ABC values of the five calibration bead populations using MS Excel. Using the linear regression, the ABCs of the beads coated with αHER2 is calculated from their MFI values. SDs are also retrieved from gated populations and transformed using linear regression. D, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. E, Normalized confluence of kidney epithelial (epithel) cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab. Each sample is normalized to its cell confluence at time zero. Graphs are identical and described in . and but with the comparison to ACTR707 instead of CAR T. F, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D and E ( n = 4). G, ELISA measurement of IFNγ levels in 24 hours supernatants of AG2G-expressing T cells or ACTR707 incubated with medium containing different concentration of IVIG ( n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. ****, P < 0.0001. Error bars represent standard error. Endothel, endothelial cells.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transformation Assay, Software, Incubation, Comparison, Concentration Assay

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Systemic administration of AG2G-expressing T cells in combination with trastuzumab eradicates HER2-expressing tumor cells in vivo . A, NCI-N87 tumor volume measured by caliper over 42 days following treatment initiation. NSG mice were injected with 2.5×10 6 NCI-N87 tumor cells, after tumor reached 70 mm 3 mice were randomized and treated once a week for 3 times (treatments are marked with arrows) with either intraperitoneal injection of saline or 250-μg trastuzumab and intravenous injection of saline or 5×10 6 AG2G-expressing T cells ( n = 6). B, Endpoint analysis of tumor volume measured by caliper, 42 days following treatment initiation. C, Endpoint analysis of tumor weight measured using scales, 42 days following treatment initiation. D, Histology images taken by x4 objective light microscopy, showing whole sections of tumors stained with H&E ( n = 6; N.D., not detected - 2 mice from AG2G+trastuzumab group). Scale bar is 5 mm. E, Representative image taken by x4 and x40 objectives using light microscopy, of one tumor from AG2G+trastuzumab group stained with anti-human CD3 antibody by IHC. F, Tumor volume measured by caliper in NSG mice treated with a single injection of T cells (10 7 ) 13 days after tumor inoculation (2.5×10 6 NCI-N87). Antibodies were injected once a week for a total of 3 times (250 μg/mouse), n = 5. G, NSG mice were injected with 1.7×10 6 NCI-N87 tumor cells, after tumor reached 100 to 200 mm 3 mice were randomized and treated once with intravenous injection of 5×10 6 AG2G-expressing T cells and with intraperitoneal injection of saline or 250-μg trastuzumab. On days 1, 7, 14, 28 following treatment, 3 mice from each group were sacrificed and RNA was extracted from blood and tumors for real-time PCR analysis of AG2G-expressing cells ( n = 12). Graphs show mean ± SD. Graphs A – C show one of two independent experiments performed. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A and B , one-way ANOVA with Holm-Sidak correction for multiple comparisons for C . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Expressing, In Vivo, Injection, Saline, Light Microscopy, Staining, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Retroviral transduction of γδ-T cells with AG2G endows them with antitumor ADCC. A, Flow cytometry analysis of sham or AG2G-transduced αβ-T cells (two left panels, activated with IL2 and anti-CD3); sham or AG2G-transduced γδ-T cells (two right panels, activated with IL2 and zoledronic acid). B, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with AG2G-expressing γδ-T cells or AG2G-expressing αβ-T cells, with or without trastuzumab ( n = 3). C, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with sham untransduced or AG2G-expressing γδ-T cells with or without trastuzumab, in E:T ratios of 1:1, 2:1 or 4:1 ( n = 4). D, ELISA measurement of IFNγ levels in supernatants of 48-hour sham or AG2G-expressing γδ-T cells cocultured with HT29-HER2 expressing cells, with or without trastuzumab (E:T ratio 4:1, n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for B and C . Two-way ANOVA with Sidak correction for multiple comparisons for D . *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: HER2 staining was performed using mouse monoclonal anti-human HER2 (Cell Signaling Technology, catalog no. 76799).

Techniques: Retroviral, Transduction, Flow Cytometry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Cancer Research

Article Title: A Wnt-Independent LGR4–EGFR Signaling Axis in Cancer Metastasis

doi: 10.1158/0008-5472.can-21-1112

Figure Lengend Snippet: Figure 4. LGR4 increases phospho-EGFR and total EGFR protein levels. A, Antibody microarray and in silico analysis identify potential targets of LGR4. B, LGR4 KO decreases phospho-EGFR and total EGFR levels in MDA231 cells upon EGF stimulation. C, Inducible expression of LGR4 enhances phospho-EGFR and total EGFR levels in MDA231 cells in the absence of recombinant EGF. D, LGR4 KO reduces levels of phospho-Src and phospho-FAK in MDA468 cells in the absence of recombinant EGF. E, Overexpression of LGR4 enhances both EGFR and HER2 levels in HCC1954 cells in the absence of recombinant EGF. Means SEMs are shown. P values were calculated by two-sided unpaired t test (B–E). n.s., not significant.

Article Snippet: Primary antibodies used in the study included LGR4 (7E7) rat antibody (37), EGFR (D38B1) XP Rabbit antibody (Cell Signaling Technology, 4276), EGFR (A-10) mouse antibody (Santa Cruz, sc373746), P-EGFR (Y1068) XP rabbit antibody (Cell Signaling Technology, 3777), MET mouse antibody (Cell Signaling Technology, 3127), HER2 (44E7) mouse antibody (Cell Signaling Technology, 2248), HA-tag mouse antibody (BioLegend 901501), b-catenin mouse antibody (BD Transduction Laboratories, 610153), Ub (P4D1) mouse antibody (Santa Cruz, sc-8017), CBL mouse antibody (Santa Cruz, sc1651), andGAPDHrabbit antibody (SantaCruz, sc-25778).

Techniques: Microarray, In Silico, Expressing, Recombinant, Over Expression

Journal: Nature Communications

Article Title: BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system

doi: 10.1038/s41467-025-59627-z

Figure Lengend Snippet: Western blot analysis of Her2 and EGFR levels in SKBR3 ( A ) or SKOV3 cell lysates ( B ) from cells treated with active or VHL Inactive BiDACs for 24 h. Panels are representative of three independent experiments. CellTiter-Glo viability analysis of SKBR3 ( C ) or SKOV3 ( D ) cells treated for 5 days with the indicated drugs. Data points show mean ± SD of three technical replicates. SKBR3, ** p = 0.0073, *** p = 0.0001. SKOV3, ** p = 0.0059, **** p < 0.0001 by two-way ANOVA, adjusted for multiple comparisons using Tukey’s multiple comparison test. E Representative fluorescence microscopy images of SKOV3 cells treated for 24 h with 1 µM of the indicated drugs. Scale bar, 20 μm. F Quantification of Her2 intensity in SKOV3 cells treated with Tuc-VHL at the indicated timepoints. Box plots show median, 25th and 75th percentiles, and whiskers show the 2nd and 98th percentiles of the distributions. **** p < 0.0001 by one-way ANOVA adjusted for multiple comparisons using Bonferroni correction. T-SNE cell morphology plots of SKOV3 ( G ) or SKBR3 ( H ) cells treated for 48 h with the indicated drugs. For T-SNE plots, dose order represents increasing drug concentrations. For ( E , F ), n = 45,000 cells were imaged at each timepoint and for each treatment condition. Source data are provided as a file.

Article Snippet: Cells were resuspended in 50 μL of a solution containing 0.5% v/v BSA in PBS, 2 μg/mL anti-EGFR Alexa Fluor 405-conjugated antibody (R&D Systems), and 2 μg/mL anti-Her2 Alexa Fluor 647-conjugated antibody (R&D Systems).

Techniques: Western Blot, Comparison, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system

doi: 10.1038/s41467-025-59627-z

Figure Lengend Snippet: T-SNE cell morphology plots of SKOV3 ( A ) or SKBR3 ( B ) cells treated for 6 h with the indicated drugs. T-SNE cell morphology plots generated without Her2 staining data for SKOV3 ( C ) or SKBR3 ( D ) cells treated for 6 h with the indicated drugs. Graphical representation of the degree of clustering between active and VHL Inactive BiDAC-dependent phenotypes in the presence ( E ) or absence ( F ) of Her2 staining information. G Representative fluorescence microscopy images of SKOV3 cells treated with the indicated drugs for 6 h. n = 45,000 cells were imaged for each treatment condition. Scale bar, 20 μm. For T-SNE plots, dose order represents increasing concentrations of drugs.

Article Snippet: Cells were resuspended in 50 μL of a solution containing 0.5% v/v BSA in PBS, 2 μg/mL anti-EGFR Alexa Fluor 405-conjugated antibody (R&D Systems), and 2 μg/mL anti-Her2 Alexa Fluor 647-conjugated antibody (R&D Systems).

Techniques: Generated, Staining, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system

doi: 10.1038/s41467-025-59627-z

Figure Lengend Snippet: A – D Flow cytometric analysis of RTK-FP levels in cells treated for 24 h with the indicated compounds. Cells in ( D ) were concurrently treated with a fixed dose of 1 µM Lap-VHL. Data points show mean ± SD of three technical replicates, and each panel is representative of two independent experiments. MFI, geometric mean fluorescence intensity. E Dot plot shows RTK-FP levels in cells treated for 24 h with 1 µM Lap-VHL. F Pseudo-color flow cytometric plots show the gates used to sort RTK-FP-positive cells treated with Lap-VHL. Enrichment of ubiquitination, neddylation and V-ATPase-related genes in sorted EGFR-RFP-positive ( G ) and Her2-GFP-positive ( H ) cells. I , J Graphical representations of protein degradation machinery identified across both RTK-FP screens. I was created in BioRender. Villa, S. (2025) https://BioRender.com/x94v608 . J was created in Biorender. Villa, S. (2025) https://BioRender.com/f36q326 . Source data are provided as a File.

Article Snippet: Cells were resuspended in 50 μL of a solution containing 0.5% v/v BSA in PBS, 2 μg/mL anti-EGFR Alexa Fluor 405-conjugated antibody (R&D Systems), and 2 μg/mL anti-Her2 Alexa Fluor 647-conjugated antibody (R&D Systems).

Techniques: Fluorescence, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system

doi: 10.1038/s41467-025-59627-z

Figure Lengend Snippet: Western blot analysis of lysates ( A ) and quantification ( B ) of untagged Her2 and EGFR-RFP levels in cells treated for 24 h with active and VHL Inactive BiDAC in combination with protein degradation inhibitors (Carfilzomib, 1 µM; MLN-7243, 200 nM; Bafilomycin-A1, 100 nM). ( A ) is representative of three biological replicates and ( B ) shows the mean ± SD of the band intensities. Control vs. Carfilzomib, ** p = 0.0016. Control vs. Bafilomycin-A1, * p = 0.0129. Control vs. MLN-7243, ** = 0.0041 by one-way ANOVA. C Western blot analysis of endogenous RTK levels in lysates from SKBR3 cells treated for 24 h with the indicated BiDACs and Bafilomycin-A1. The blot shows the results of a single experiment. D – I Flow cytometric analysis of RTK-FP levels in cells treated for 8 h with the indicated BiDACs and protein degradation inhibitors (HCQ hydroxychloroquine, 30 µM). Data points show mean ± SD of three technical replicates, and each panel is representative of two independent experiments. D *** p = 0.0001. F ** p = 0.0012. H *** p = 0.0004. I ** p = 0.0019. D – I **** p < 0.0001 by-two ANOVA. MFI, geometric mean fluorescence intensity. J Confocal microscopy of RTK-FP cells expressing LAMP1-SNAP treated for 8 h with 1 µM Lap-VHL alone or in combination with 100 nM Bafilomycin-A1. Images are representative of n = 100 cells. Scale bar, 20 μm. K Histogram of colocalization scores between LAMP1-SNAP-positive lysosomes and EGFR-RFP in cells imaged in ( J ). n = 10,000 lysosomes were identified for colocalization analysis of each treatment condition. L Flow cytometric analysis of RTK-FP levels in cells treated for 8 h with epidermal growth factor (EGF) and protein degradation inhibitors, as in ( D – I ). Data points show mean ± SD of three technical replicates. **** p < 0.0001 by two-way ANOVA. For all combination treatments, cells were pretreated for 30 min with degradation inhibitors prior to addition of BiDACs. P -values calculated by ANOVA were adjusted using Tukey’s multiple comparisons test. Source data are provided as a file.

Article Snippet: Cells were resuspended in 50 μL of a solution containing 0.5% v/v BSA in PBS, 2 μg/mL anti-EGFR Alexa Fluor 405-conjugated antibody (R&D Systems), and 2 μg/mL anti-Her2 Alexa Fluor 647-conjugated antibody (R&D Systems).

Techniques: Western Blot, Control, Fluorescence, Confocal Microscopy, Expressing

Journal: Nature Communications

Article Title: BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system

doi: 10.1038/s41467-025-59627-z

Figure Lengend Snippet: A Flow cytometric analysis of surface RTK levels in cells treated for 24 h with 1 µM Lap-VHL. Flow cytometric analysis of surface EGFR ( B , E ) or surface Her2 ( C , D ) levels in cells treated for 24 h with the indicated BiDACs or EGF in combination with protein degradation inhibitors (Carfilzomib, 1 µM; MLN-7243, 200 nM; Bafilomycin-A1, 100 nM). Data points show mean ± SD of three technical replicates, and each panel is representative of two independent experiments. **** p < 0.0001; ns not significant by two-way ANOVA. MFI, geometric mean fluorescence intensity. F Confocal timelapse microscopy of RTK-FP cells treated with 1 µM Lap-VHL or Lap-VHL Inactive in combination with Bafiloymcin-A1 or Carfilzomib. Images at T = 0 h were captured immediately after addition of BiDACs. Montages are representative of 28 fields imaged for each condition. Scale bar, 20 μm. Quantification of the number of Her2-GFP-positive ( G ) or EGFR-RFP-positive ( H ) puncta in cells treated with Lap-VHL and the indicated protein degradation inhibitors. Each data point shows the mean ± SD of three replicate wells, and the results are representative of two independent experiments. I , J RTK-FP cells were treated with 1 μM BiDACs alone or together with 1 μM Carfilzomib for 3 h. Her2-GFP was immunopurified and levels of Her2-GFP-associated K48-ubiquitin were analyzed by western blot. The graph shows the mean ± SD fold change in the K48-ubiquitin signal in the affinity purifications, which was normalized to levels of purified Her2-GFP. The data was obtained from three independent experiments. ** p = 0.0083, * p = 0.0248, ns not significant by one-way ANOVA. For ( B – H ), cells were pretreated for 30 min with degradation inhibitors prior to addition of BiDACs. P -values calculated by ANOVA were adjusted using Tukey’s multiple comparisons test. Source data are provided as a file.

Article Snippet: Cells were resuspended in 50 μL of a solution containing 0.5% v/v BSA in PBS, 2 μg/mL anti-EGFR Alexa Fluor 405-conjugated antibody (R&D Systems), and 2 μg/mL anti-Her2 Alexa Fluor 647-conjugated antibody (R&D Systems).

Techniques: Fluorescence, Microscopy, Ubiquitin Proteomics, Western Blot, Purification

Journal: Nature Communications

Article Title: BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system

doi: 10.1038/s41467-025-59627-z

Figure Lengend Snippet: A Control and PQLC2-targeting sgRNA counts from Lap-VHL-treated RTK-FP cells sorted for high EGFR-RFP levels, compared to those from unsorted cells. B Genomic PCR analysis of the fraction of frameshifted PQLC2-coding alleles in cell pools expressing Cas9 and sgRNAs targeting PQLC2. Histograms (1 µM Lap-VHL) ( C ) and quantification ( D ) of EGFR-RFP levels in control or clonal PQLC2 KO cells treated for 24 h with Lap-VHL. Control vs. Clone #1, *** p = 0.0009. Control vs. Clone #2, *** p = 0.0003. ns, not significant by two-way ANOVA adjusted using Tukey’s multiple comparison test. Each data point shows a single measurement, and the panel is representative of two independent experiments. Histograms (10 ng/ml EGF) ( E ) and quantification ( F ) of EGFR-RFP levels in control or PQLC2 KO cells treated for 24 h with EGF. **** p < 0.0001 by two-way ANOVA. Data points in ( F ) show the mean ± SD of three technical replicates, and the panel is representative of two independent experiments. G Confocal microscopy of control or PQLC2 KO cells expressing LAMP1-SNAP, treated for 24 h with 1 μM Lap-VHL or Lap-VHL Inactive . Scale bar, 20 μm. H Distribution of EGFR-RFP puncta by size in n = 10 fields imaged in ( G ). I RTK-FP cells were pre-treated with 100 nM Bafilomycin-A1 for 30 min and subsequently treated with 1 μM Lap-VHL. The number of Her2-GFP puncta at each time point, shown as mean ± SD, was quantified using time lapse microscopy, and the rate of accumulation of Her2-GFP puncta was determined by fitting the data using linear regression analysis. ns, slopes not significantly different ( p = 0.1019) using multiple regression analysis with dummy coded variables (GraphPad Prism). n = 18 fields were analyzed for each condition. For ( C – I ), RTK-FP control cells express an sgRNA targeting the cell surface protein CD81. Source data are provided as a file.

Article Snippet: Cells were resuspended in 50 μL of a solution containing 0.5% v/v BSA in PBS, 2 μg/mL anti-EGFR Alexa Fluor 405-conjugated antibody (R&D Systems), and 2 μg/mL anti-Her2 Alexa Fluor 647-conjugated antibody (R&D Systems).

Techniques: Control, Expressing, Comparison, Confocal Microscopy, Time-lapse Microscopy

Journal: Endocrinology

Article Title: Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

doi: 10.1210/endocr/bqae129

Figure Lengend Snippet: Figure 5. Ovaries were collected from pregnant rats on day 12 for immunohistochemistry (IHC) (n = 3 rats per group). A, Representative photomicrographs view of corpus luteum (CL) with immunolocalization of epidermal growth factor receptor 3 (ErbB3) and steroidogenic acute regulatory protein (StAR) with Alexa Fluor 594– (red) and Alexa Fluor 488– (green) labeled secondary antibodies, respectively. The nucleus was stained with DAPI (4′,6′-diamidino-2-phenylindole) (blue). B. Representative photomicrographs view of CL with immunolocalization of ErbB2 and StAR with Alexa Fluor 594– (red) and Alexa Fluor 488– (green) labeled secondary antibodies, respectively. The nucleus was stained with DAPI (blue). C, Representative photomicrographs view of CL with immunolocalization of ErbB3 and ErbB2 with Alexa Fluor 594– (red) and Alexa Fluor 488– (green) labeled secondary antibodies, respectively. The nucleus was stained with DAPI (blue).

Article Snippet: Scientific Mouse (monoclonal) AB_2816212 http://antibodyregistry.org/AB_2816212 1:500 Total ErbB2 Anti-ErbB2 Origene Mouse (monoclonal) AB_2629050 http://antibodyregistry.org/AB_2629050 1:500 (WB) 1:50 (IHC)

Techniques: Immunohistochemistry, Labeling, Staining

Journal: Endocrinology

Article Title: Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

doi: 10.1210/endocr/bqae129

Figure Lengend Snippet: Figure 6. Effects of knockdown of endogenous neuregulin 1 (NRG1) on the exogenous treatment of tumor necrosis factor α (TNFα) on phospho(p)- and total ErbB2, ErbB3, phosphoinositide 3-kinase (PI3K), serine/threonine-protein kinase (AKT/protein kinase B), and extracellular signal-regulated kinase (ERK1/2) protein expressions in rat luteal cells (LCs). LCs were grown at 70% to 80% confluency and transiently transfected with small interfering-NRG1 (siNRG1) or scrambled RNA. After that, cells were maintained in serum-starved media and treated with TNFα at different concentrations (10 and 50 ng/mL) for 24 hours. A and D, Expression of phospho(p)- and total ErbB2, ErbB3 PI3K, AKT/PKB, and ERK1/2 protein expressions were determined by Western blot analysis. To each lane of the gel equal amounts of protein were applied. β-Actin was used as an internal control. Bar diagrams B to F represent the densitometric protein analyses in immunoblots (WBs). All bar graphs represent the mean ± SEM of results from 3 individual experiments (n = 3). Asterisks represent unpaired t test, *P less than or equal to .01; **P less than or equal to .001; ***P less than or equal to .0001; and NS, not significant.

Article Snippet: Scientific Mouse (monoclonal) AB_2816212 http://antibodyregistry.org/AB_2816212 1:500 Total ErbB2 Anti-ErbB2 Origene Mouse (monoclonal) AB_2629050 http://antibodyregistry.org/AB_2629050 1:500 (WB) 1:50 (IHC)

Techniques: Knockdown, Transfection, Expressing, Western Blot, Control