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: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: The expression of human HER2 in murine liver by recombinant adenovirus delivery. ( A ) Design of the adenovirus vector carrying a truncated human HER2/ERBB2 ( tHER2 ) gene and a firefly luciferase gene, whose expression are driven by the CMV and SV40 promoter, respectively. ( B ) In vitro testing luciferase and HER2/ERBB2 expression in B16 cell (the murine melanoma cell line). A two-tailed unpaired t -test of luciferase and HER2/ERBB2 level was used for statistical analysis. ( C ) In vivo imaging of luciferase expression in mice injected with 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 8 days post-infection (8dpi). ( D ) IHC staining of liver section from mice that received different dosage of Ad5-HER2 for human HER2 (brown). ( E ) Quantification of HER2 in the indicated liver tissue. Data presented as mean ± SEM, each group contained 4 mice. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Recombinant, Plasmid Preparation, Luciferase, In Vitro, Two Tailed Test, In Vivo Imaging, Injection, Infection, Immunohistochemistry

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: CAR-T cells are activated by human antigen and accumulated in murine liver. Human HER2 was expressed in murine liver following 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 adenovirus delivery. Mice were then received either 5E + 6 HER2 CAR-T cells or UTD. ( A ) Systemic cytokine secretion by T cells was determined in murine serum (n = 3–4) by cytometric beads array 5 days after T cells transfer. ( B ) Frequency of HER2 CAR-T cells (up panel) and HER2 CAR-T cell counts (down panel) among live nucleated cells in the indicated tissues 7 days after T cells infusion. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques:

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: HER2 CAR-T cells cause acute on-target liver toxicity in mice. ( A ) Flow chart of the experimental design for evaluating on-target, liver injury of CAR-T cells. NSG mice received 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 and then infused with either 5E + 6 HER2 CAR-T cells or untransduced T cells (UTD) 8dpi. An n = 3–4 mice per group is shown in each panel unless stated otherwise. ( B ) Weight change shown by percent change from initial weight ± SEM in mice that received different dosage of Ad5-HER2 and then either HER2 CAR-T cells or UTD. ( C ) Survival curves of NSG mice that received different dosage of Ad5-HER2 and then T cells transfer. Statistical analysis was performed using a log-rank Mantel-Cox test. ( D ) Liver function profile as determined by serum ALT and AST levels collected days as indicated after T cells infusion. Mean ALT/AST ± SEM in mice received different dosage of Ad5-HER2 and then T cells injection. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Injection

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: Hypoxia-response CAR-T cells don’t cause acute on-target liver toxicity in mice expressing high levels of human antigen. ( A ) Flow chart of the experimental design for evaluating on-target, liver injury of CAR-T cells. NSG mice received 1E + 9 PFU of Ad5-HER2 and then infused with either 5E + 6 HER2 CAR-T cells or hypoxia-response CAR-T cells. A human antigen negative control group of NSG mice received 5E + 6 HER2 CAR-T cells but no Ad5-HER2 (i.e. Ad-). An n = 3–4 mice per group is shown in each panel unless stated otherwise. ( B ) Weight change shown by percent change from initial weight ± SEM in mice that received 1E + 9 PFU of Ad5-HER2 and then either CAR-T cells or hypoxia-response CAR-T cells. ( C ) Survival curves of NSG mice that received 1E + 9 PFU of Ad5-HER2 and then T cells transfer. Statistical analysis was performed using a log-rank Mantel-Cox test. ( D ) Liver function profile as determined by serum ALT levels collected days as indicated after T cells infusion. Mean ALT ± SEM in mice (n = 3–4) received 1E + 9 PFU of Ad5-HER2 and then T cells injection. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance between UTD group and other groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Negative Control, Injection

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: Hypoxia-response CAR-T cells are not activated and accumulated by human antigen in murine liver. Human HER2 was expressed in murine liver following 1E + 9 PFU of Ad5-HER2 delivery. Mice were then received 5E + 6 HER2 CAR-T cells or hypoxia-response HER2 CAR-T cells. Control groups included mice that had hepatic HER2 expression but untransduced T cells (UTD) and mice that received HER2 CAR-T cells but lacked hepatic HER2 expression (i.e. Ad-). ( A ) Systemic cytokine secretion by T cells was determined in murine serum (n = 4–13) by cytometric beads array 7 days after T cells transfer. ( B ) Quantitation of inflammatory infiltration lesions in the liver 7 days after T cells transfer. ( C ) Frequency of CAR-T cells (up panel) and CAR-T cell counts (down panel) among live nucleated cells in the indicated tissues 7 days after T cells infusion. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Quantitation Assay

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 1. Expression of the scFv and scFv-CD40 CRs in murine DC. A, schematic representation of the scFv and scFv-CD40 CRs. Each construct consisted of the ErbB2-specific scFv, a c-myc tag, the hinge region of murine CD8a, and the transmembrane (TM) and/or cytoplasmic regions of murine CD40 signaling chain. B, after 5 d of DC differentiation, surface expression of MHC and costimulatory molecules in immature DC was assessed by immunofluorescent staining with antibodies specific for CD40, CD86, MHC class I Kd, and MHC class II I-Ad along with anti-CD11c antibody. DC treated with 1 Ag/mL LPS for 24 h were included as a positive control. C, analysis of expression of either GFP or CR expression in DC after adenoviral infection at 100 MOI. Top, GFP expression in uninfected DC or GFP-DC; bottom, CR expression in GFP-DC, scFv-DC, or scFv-CD40-DC. D, lysates of GFP-DC (lanes 1 and 4), scFv-DC (lanes 2 and 5), and scFv-CD40-DC (lanes 3 and 6) were separated by SDS-PAGE under reducing (R) or nonreducing (NR) conditions. Immunoblot analysis was performed with an anti–c-myc or anti–a-tubulin mAb followed by chemiluminescent detection. The position of molecular weigh standards (kd) is indicated.

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: Expressing, Construct, Staining, Positive Control, Infection, SDS Page, Western Blot

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 2. The scFv-CD40 CR induced NF-nB–dependent DC activation and effector function after ErbB2 stimulation. A, NF-nB mRNA levels from GFP-DC, scFv-DC, or scFv-CD40-DC stimulated by 1 Ag/mL BSA or ErbB2 protein for 24 h were detected by real-time PCR. Results are normalized to glyceraldehyde-3-phosphate dehydrogenase and expressed as fold increase relative to uninfected DC. B, nuclear lysates from infected DC stimulated by titrated levels of ErbB2 protein (1, 100, and 1000 ng/mL) were prepared for RelB/Sp1-specific Western blots. Infected DC stimulated by 1 Ag/mL LPS was included as a positive control. C, l, infected DC were cultured with the irradiated D2F2 or D2F2/E2 tumor cells at a DC to tumor ratio of 5:1 at 37jC. After 24 h, DC were analyzed for the expression of costimulatory and MHC molecules by flow cytometry. Results are normalized to uninfected-DC MFI. II, infected DC were treated as described in I before supernatants were collected and cytokine levels were determined by ELISA. D, l, infected DC were incubated with the D2F2 or D2F2/E2 tumor cells at a DC to tumor ratio of 5:1 at 37jC for 24 h and cytotoxicity was evaluated by MTT assay. Wells with medium alone were used as background control (BG) and wells for total viability/spontaneous death of untreated cells (TS) contained only medium and tumor cells. Experimental (Ex) wells contained tumor cells and infected DC. Percentage of cytotoxicity was calculated using the following formula: % cytotoxicity = [(TS BG) (Ex BG)]/(TSBG) 100%. II, D2F2 or D2F2/E2 tumor cells were stained with 4 Ag/mL Hoechst 33342 for 30 min. Cells were washed and maintained in culture for additional 3 h before washing again and adding infected DC at a DC to tumor ratio of 5:1 at 37jC or 4jC. After 24 h, the cells were harvested and stained with anti-mouse CD11c mAb and PI before flow cytometry analysis. Viable DC (CD11c+PI) were gated for analysis, and MFI of Hoechst 33342 staining is reported. All results are representative of three independent experiments. *, P < 0.05; **, P < 0.01; NF-nB mRNA, costimulatory, and MHC molecules expression, cytokine levels, or tumoricidal activity significantly increased in scFv-CD40-DC after ErbB2 ligation compared with the controls (GFP-DC or scFv-DC).

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Infection, Western Blot, Positive Control, Cell Culture, Irradiation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Control, Staining, Activity Assay, Ligation

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 4. In vivo antitumor effects of the CR-modified DC. A, D2F2/E2 tumors. BALB/c mice were injected s.c. in the left flank on day 0 with 2 105 ErbB2-positive D2F2/E2 mammary tumor cells. On day 8 and 11, 5 105 scFv-DC or scFv-CD40-DC were injected i.v. B, D2F2 tumors. BALB/c mice were injected s.c. on day 0 with 2 105 ErbB2-negative D2F2 mammary tumor cells. On day 8 and 11, 5 105 scFv-DC, scFv-CD40-DC was injected i.v. C, EL4/E2 tumors. C57BL/6 mice were injected s.c. on day 0 with 2 105 ErbB2-positive EL4/E2 thymoma cells. On day 8 and 11, 5 105 scFv-DC or scFv-CD40-DC were injected i.v. D, EL4 tumors. C57BL/6 mice were injected s.c. on day 0 with 2 105 ErbB2-negative EL4 thymoma cells. On day 8 and 11, 5 105 scFv-DC or scFv-CD40-DC was injected i.v. In all tumor models, mice injected with PBS were used as an additional control. Tumor size was measured twice weekly and survival was monitored for 12 wk. *, P < 0.05; **, P < 0.01, tumor growth was significantly inhibited in the scFv-CD40-DC–treated group compared with the scFv-DC or PBS treatment group.

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: In Vivo, Modification, Injection, Control

Journal: Cancer Research

Article Title: Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

doi: 10.1158/0008-5472.can-07-6051

Figure Lengend Snippet: Figure 5. Induction of tumor-specific CTLs after i.v. administration of scFv- CD40-DC. Mice were implanted s.c. in the left flank on day 0 with 2 105 ErbB2- positive D2F2/E2 mammary tumor cells. On day 8 and 11, 5 105 scFv-DC, scFv-CD40-DC, or PBS alone was injected i.v. Ten days after treatment, the splenocytes were isolated and restimulated in vitro for 5 d with the irradiated D2F2/E2 cells and assayed for CTL activity against 51Cr-labeled D2F2/E2 (A), D2F2 (B), and NIH3T3 targets (C) or D2F2/E2 targets in the presence of neutralizing or isotype-matched control mAb (10 Ag/mL) as indicated (D). Points, the mean of triplicate cultures, representative of three independent experiments; bars, SE.

Article Snippet: For nuclear Rel-B blotting, infected DC were stimulated in 24-well plates (106 cells per well) by various concentrations of immobilized bovine serum albumin (BSA), recombinant ErbB2 protein (R&D Systems), or 1 Ag/mL LPS (SigmaAldrich) for 24 h. Nuclear lysates were prepared using the Transfactor Extraction kit (Clontech Laboratories) and probed with mouse Rel-B– and Sp1-specific mAb (Santa Cruz Biotechnology).

Techniques: Injection, Isolation, In Vitro, Irradiation, Activity Assay, Labeling, Control