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R&D Systems goat polyclonal antibody against human cxcl12
Sequences of primers used in this study

Goat Polyclonal Antibody Against Human Cxcl12, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/system+310/Human+CXCL12%2FSDF-1+Antibody/pmc04063227-43-1-7
Average 93 stars, based on 34 article reviews

goat polyclonal antibody against human cxcl12 - by Bioz Stars, 2026-10

93/100 stars

Images

1) Product Images from "COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration"

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-407

Figure Legend Snippet: Sequences of primers used in this study

Techniques Used:

COUP-TFI modifies the expression of the CXCL12 signaling axis in MCF-7 cells. (A) Characterization of the control and COUP clones. An immunofluorescence cytochemistry assay was used to detect the relative expression of HA/COUP-TFI or COUP-TFI proteins in the control (Cont.) and COUP clones. The cells were fixed and processed for immunofluorescence as described in Methods; the nuclei were stained with DAPI. (B) CXCL12 , CXCR4 , and CXCR7 mRNAs were quantified by a real-time PCR analysis from two independent MCF-7 control and COUP clones. The results were normalized to GAPDH mRNA used as an internal control. The results were expressed as the relative mRNA expression level of CXCL12 , CXCR4 , or CXCR7 . Data are the mean values ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (C) The amount of intracellular HA/COUP-TFI, COUP-TFI, CXCL12, CXCR4, and CXCR7 protein was determined from whole-cell extracts of the different MCF-7 clones and compared to total ERK. A representative western blot is shown. (D) The control and COUP clones were fixed, and an immunofluorescence cytochemistry assay was used to detect the relative expression of CXCL12, CXCR4, and CXCR7 proteins. Staining with DAPI is also presented to visualize the nucleus of the cells.

Figure Legend Snippet: COUP-TFI modifies the expression of the CXCL12 signaling axis in MCF-7 cells. (A) Characterization of the control and COUP clones. An immunofluorescence cytochemistry assay was used to detect the relative expression of HA/COUP-TFI or COUP-TFI proteins in the control (Cont.) and COUP clones. The cells were fixed and processed for immunofluorescence as described in Methods; the nuclei were stained with DAPI. (B) CXCL12 , CXCR4 , and CXCR7 mRNAs were quantified by a real-time PCR analysis from two independent MCF-7 control and COUP clones. The results were normalized to GAPDH mRNA used as an internal control. The results were expressed as the relative mRNA expression level of CXCL12 , CXCR4 , or CXCR7 . Data are the mean values ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (C) The amount of intracellular HA/COUP-TFI, COUP-TFI, CXCL12, CXCR4, and CXCR7 protein was determined from whole-cell extracts of the different MCF-7 clones and compared to total ERK. A representative western blot is shown. (D) The control and COUP clones were fixed, and an immunofluorescence cytochemistry assay was used to detect the relative expression of CXCL12, CXCR4, and CXCR7 proteins. Staining with DAPI is also presented to visualize the nucleus of the cells.

Techniques Used: Expressing, Control, Clone Assay, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot

COUP-TFI modulates the chromatin structure of the CXCL12 and CXCR4 gene promoters. The FAIRE assay was performed as described in Methods. Real-time PCR was performed to monitor the enrichment of DNA corresponding to the proximal promoter of the CXCL12 (A) , the CXCR4 (B) and the CXCR7 (C) genes relative to the input chromatin from the control (Cont.) and COUP clones. The data are from triplicate samples and are representative of three separate experiments. The asterisk indicates significant differences ( p < 0.05) between the control and COUP clones.

Figure Legend Snippet: COUP-TFI modulates the chromatin structure of the CXCL12 and CXCR4 gene promoters. The FAIRE assay was performed as described in Methods. Real-time PCR was performed to monitor the enrichment of DNA corresponding to the proximal promoter of the CXCL12 (A) , the CXCR4 (B) and the CXCR7 (C) genes relative to the input chromatin from the control (Cont.) and COUP clones. The data are from triplicate samples and are representative of three separate experiments. The asterisk indicates significant differences ( p < 0.05) between the control and COUP clones.

Techniques Used: Real-time Polymerase Chain Reaction, Control, Clone Assay

Estrogenic regulation of CXCL12 and CXCR4 in control and COUP clones. Control (Cont.) and COUP clones were treated with ethanol (EtOH) as the vehicle or E2 10 −8 M and ICI 10 −6 M alone or both together for 48 h. The CXCL12 (A) and CXCR4 (B) relative mRNA levels were monitored by a real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Figure Legend Snippet: Estrogenic regulation of CXCL12 and CXCR4 in control and COUP clones. Control (Cont.) and COUP clones were treated with ethanol (EtOH) as the vehicle or E2 10 −8 M and ICI 10 −6 M alone or both together for 48 h. The CXCL12 (A) and CXCR4 (B) relative mRNA levels were monitored by a real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Techniques Used: Control, Clone Assay, Real-time Polymerase Chain Reaction, Expressing

The effect of COUP-TFI on the CXCL12/CXCR4 axis is mediated by EGF/EGFR activation. (A) The relative expression of EGF and EGFR mRNA was monitored by a real-time PCR analysis using MCF-7 control (Cont.) and COUP clones. The results were normalized against GAPDH as the internal control and are expressed as the mean EGF or EGFR mRNA/GAPDH mRNA ratio ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (B) ERK activation was examined in the MCF-7 control (Cont.) and COUP clones after a 5- or 10-min stimulation with EGF (10 −9 M) or CXCL12 (200 ng/mL). Western blots were performed using antibodies against phospho-ERK (P-ERK) and total ERK (ERK1/2); a representative western blot is presented. The importance of EGFR-specific signaling and general ERK signaling on CXCL12 (C) and CXCR4 (D) regulation was assayed by treating the cells with EGF (10 −9 M), AG1478 (25 μM), or U0126 (25 μM) for 48 h. The CXCL12 and CXCR4 relative mRNA levels were monitored by the real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Figure Legend Snippet: The effect of COUP-TFI on the CXCL12/CXCR4 axis is mediated by EGF/EGFR activation. (A) The relative expression of EGF and EGFR mRNA was monitored by a real-time PCR analysis using MCF-7 control (Cont.) and COUP clones. The results were normalized against GAPDH as the internal control and are expressed as the mean EGF or EGFR mRNA/GAPDH mRNA ratio ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (B) ERK activation was examined in the MCF-7 control (Cont.) and COUP clones after a 5- or 10-min stimulation with EGF (10 −9 M) or CXCL12 (200 ng/mL). Western blots were performed using antibodies against phospho-ERK (P-ERK) and total ERK (ERK1/2); a representative western blot is presented. The importance of EGFR-specific signaling and general ERK signaling on CXCL12 (C) and CXCR4 (D) regulation was assayed by treating the cells with EGF (10 −9 M), AG1478 (25 μM), or U0126 (25 μM) for 48 h. The CXCL12 and CXCR4 relative mRNA levels were monitored by the real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Clone Assay, Western Blot

COUP-TFI overexpression influences cellular responses to CXCL12. (A) The relative growth of the control (Cont.) and COUP clones was assayed with or without CXCL12 treatment for 7 days. The basal and CXCL12-induced cell growth were evaluated by MTT assays (n = 6) and determined in three independent experiments. The results are expressed as the relative cell number obtained when the control cells were treated with the vehicle control. Significant differences between the unstimulated control cells and the other conditions ( p < 0.05) are indicated with an asterisk. Significant differences between stimulated control cells and stimulated COUP clones ( p < 0.05 ) are indicated with a pound sign. (B) The migratory capacity of control (Cont.) and COUP clones was analyzed. The cells were maintained in phenol red-free DMEM/2.5% dsFBS for 48 h and then seeded in phenol red-free DMEM/0.5% dsFBS in the upper chamber of a PET 8-μm pore insert. The cells were allowed to migrate for 24 h toward the phenol red-free DMEM/2.5% dsFBS medium complemented or not with CXCL12 (200 ng/mL) and AMD3100 (50 μM). (C) CXCL12 was also added to the culture medium in the upper chamber prior to migration. The results are expressed as the mean ± SEM of the relative number of migratory cells compared to the basal migration of the control cells measured in three independent experiments. The asterisks indicate significant differences ( p < 0.05) from the basal migration of the control clones. The pound sign indicates significant differences ( p < 0.05) between two conditions linked by black lines.

Figure Legend Snippet: COUP-TFI overexpression influences cellular responses to CXCL12. (A) The relative growth of the control (Cont.) and COUP clones was assayed with or without CXCL12 treatment for 7 days. The basal and CXCL12-induced cell growth were evaluated by MTT assays (n = 6) and determined in three independent experiments. The results are expressed as the relative cell number obtained when the control cells were treated with the vehicle control. Significant differences between the unstimulated control cells and the other conditions ( p < 0.05) are indicated with an asterisk. Significant differences between stimulated control cells and stimulated COUP clones ( p < 0.05 ) are indicated with a pound sign. (B) The migratory capacity of control (Cont.) and COUP clones was analyzed. The cells were maintained in phenol red-free DMEM/2.5% dsFBS for 48 h and then seeded in phenol red-free DMEM/0.5% dsFBS in the upper chamber of a PET 8-μm pore insert. The cells were allowed to migrate for 24 h toward the phenol red-free DMEM/2.5% dsFBS medium complemented or not with CXCL12 (200 ng/mL) and AMD3100 (50 μM). (C) CXCL12 was also added to the culture medium in the upper chamber prior to migration. The results are expressed as the mean ± SEM of the relative number of migratory cells compared to the basal migration of the control cells measured in three independent experiments. The asterisks indicate significant differences ( p < 0.05) from the basal migration of the control clones. The pound sign indicates significant differences ( p < 0.05) between two conditions linked by black lines.

Techniques Used: Over Expression, Control, Clone Assay, Migration

Box plots of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA expression in breast cancer and normal tissue. CXCR4 (A) , CXCR7 (B) , CXCL12 (C) , and COUP-TFI (D) mRNA expression was measured by real-time PCR in 23 normal breast tissue samples (NT), in 20 SBR grades 1 and 2, and in 19 SBR grades 3. The expression level was normalized by 18S RNA expression and analyzed with IQ5 software (Bio-Rad). The data are presented as whisker plots in which the horizontal bar represents the median, the grey boxes are the 25 th and 75 th percentiles, the vertical bar is the standard deviation, and the plus signs are the extreme points. All the Mann-Whitney tests were performed with Minitab 16 software, and the p value is indicated on the different graphs (ns denotes non-significant).

Figure Legend Snippet: Box plots of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA expression in breast cancer and normal tissue. CXCR4 (A) , CXCR7 (B) , CXCL12 (C) , and COUP-TFI (D) mRNA expression was measured by real-time PCR in 23 normal breast tissue samples (NT), in 20 SBR grades 1 and 2, and in 19 SBR grades 3. The expression level was normalized by 18S RNA expression and analyzed with IQ5 software (Bio-Rad). The data are presented as whisker plots in which the horizontal bar represents the median, the grey boxes are the 25 th and 75 th percentiles, the vertical bar is the standard deviation, and the plus signs are the extreme points. All the Mann-Whitney tests were performed with Minitab 16 software, and the p value is indicated on the different graphs (ns denotes non-significant).

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Software, Whisker Assay, Standard Deviation, MANN-WHITNEY

Recombinant:

Article Title: The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD + cells via RUNX1.
Article Snippet: .. Recombinant murine and human CXCL12 and PE-conjugated antihuman/mouse CXCR7 antibodies were purchased from R&D Systems (Minneapolis, MN). .. PE-conjugated anti-RUNX1 antibody and rat IgG2a isotype were purchased from eBioscience (San Diego, CA).

Incubation:

Article Title: The acidic intrinsically disordered region of the inflammatory mediator HMGB1 mediates fuzzy interactions with CXCL12
Article Snippet: .. Cells were then incubated overnight at 4 °C with two primary antibodies: mouse monoclonal anti-HMGB1 (1:1000; HMGBiotech, HM-901) and goat polyclonal anti-CXCL12 (1:50; R&D Systems, #AF-310-NA). .. Following three washes with 0.2% BSA/PBS, cells were incubated with secondary oligonucleotide-linked antibodies for 1 h at 37 °C (anti-mouse PLUS [#DUO92001] and anti-goat MINUS [#DUO92006], Duolink, Sigma-Aldrich; 1:5 dilution in Duolink antibody diluent [DUO82008]) and processed according to the Duolink PLA fluorescence protocol (Sigma).

Western Blot:

Article Title: Estrogen receptor α promotes lung cancer cell invasion via increase of and cross-talk with infiltrated macrophages through the CCL2/CCR2/MMP9 and CXCL12/CXCR4 signaling pathways.
Article Snippet: The anti-mouse ERa (E115) for western blot was from Abcam. .. The CXCL12 (AF-310-NA) antibody for western blot was from R&D Systems (Minneapolis, MN, USA). .. Antimouse/anti-rabbit secondary antibody for western blot was from Invitrogen (Carlsbad, CA, USA).

Image Search Results

EGFRvIII expression in WT HNSCC cell lines. A Schematic structure of EGFR and EGFRvIII. The EGFRvIII variant is characterized by an in-frame deletion of exons 2–7 in the full-length EGFR gene. The domains of EGFR are shown, labeled domains L1 (also known as domain 1), CR1 (domain 2), L2 (domain 3) and CR2 (domain 4), respectively (adapted from Gan et al. 2009 ). B RT-PCR analysis of EGFRvIII mRNA expression. The presence of EGFRvIII mRNA was assessed in HNSCC cell lines by RT-PCR. A representative ethidium bromide-stained agarose gel shows PCR products for EGFR (1037 bp) and EGFRvIII (236 bp). Primary human fibroblasts (Fibro) served as negative control for EGFRvIII, while DK-MG cells were used as a positive control. Multiple HNSCC cell lines were analyzed. C and D Flow cytometric analysis of EGFRvIII protein expression. Cell lines were stained using a recombinant rabbit monoclonal EGFRvIII antibody followed by a PE-conjugated anti-rabbit IgG (H + L) F(ab’)₂ secondary antibody. EGFR expression was detected using APC- or PE-conjugated human EGFR antibodies. C Relative mean fluorescence intensity (MFI; y-axis) of EGFRvIII and EGFR expression in HNSCC cell lines. EGFRvIII data represent the mean ± SEM of three independent experiments. D Representative histograms of EGFRvIII (red), EGFR (blue), isotype staining with secondary antibody or isotype alone (dark green), and unstained cells (black). Normalized cell count is plotted against the fluorescence intensity

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: EGFRvIII expression in WT HNSCC cell lines. A Schematic structure of EGFR and EGFRvIII. The EGFRvIII variant is characterized by an in-frame deletion of exons 2–7 in the full-length EGFR gene. The domains of EGFR are shown, labeled domains L1 (also known as domain 1), CR1 (domain 2), L2 (domain 3) and CR2 (domain 4), respectively (adapted from Gan et al. 2009 ). B RT-PCR analysis of EGFRvIII mRNA expression. The presence of EGFRvIII mRNA was assessed in HNSCC cell lines by RT-PCR. A representative ethidium bromide-stained agarose gel shows PCR products for EGFR (1037 bp) and EGFRvIII (236 bp). Primary human fibroblasts (Fibro) served as negative control for EGFRvIII, while DK-MG cells were used as a positive control. Multiple HNSCC cell lines were analyzed. C and D Flow cytometric analysis of EGFRvIII protein expression. Cell lines were stained using a recombinant rabbit monoclonal EGFRvIII antibody followed by a PE-conjugated anti-rabbit IgG (H + L) F(ab’)₂ secondary antibody. EGFR expression was detected using APC- or PE-conjugated human EGFR antibodies. C Relative mean fluorescence intensity (MFI; y-axis) of EGFRvIII and EGFR expression in HNSCC cell lines. EGFRvIII data represent the mean ± SEM of three independent experiments. D Representative histograms of EGFRvIII (red), EGFR (blue), isotype staining with secondary antibody or isotype alone (dark green), and unstained cells (black). Normalized cell count is plotted against the fluorescence intensity

Article Snippet: For sorting of EGFRvIII-positive cells, 5 × 10 6 cells were stained with a PE-conjugated EGFR antibody (clone REA688; Miltenyi Biotec; PE: 130-110-528), which recognizes an EGFR epitope retained in EGFRvIII.

Techniques: Expressing, Variant Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, Analysis, Staining, Agarose Gel Electrophoresis, Negative Control, Positive Control, Recombinant, Fluorescence, Cell Characterization

Artificial overexpression of EGFRvIII at different levels in HNSCC cell lines. A Schematic representation of the lentiviral EGFRvIII expression vector. The internal promoters (iP: hPGK, hEF1α and SFFV) drive expression of the EGFRvIII transgene and via an IRES site also the Geneticin resistance gene nptII (neoR). B - D UM-14C KO and UT-24A KO cells were transduced with LV EGFRvIII expression vectors. EGFRvIII-positive EGFR KO cells were further purified by flow sorting, generating new EGFRvIII-expressing cell lines. B and C Flow cytometric analysis of EGFRvIII surface expression after sorting. UM-14C and UT-24A cells were stained with ( B ) a PE-conjugated EGFR antibody for EGFR and EGFRvIII expression. Left: EGFR expression is shown by histogram. Right: Means ± SEM of MFI values of EGFRvIII-positive EGFR KO UM-14C and UT-24A cells as well as the corresponding UM-14C and UT-24A KO, WT and control cells transduced with an empty vector (data from three independent experiments) are shown. Differences between promoter groups within each cell line were analyzed using one-way repeated-measures ANOVA followed by Tukey’s multiple-comparisons test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); ns, not significant. C EGFRvIII expression was additionally determined using an EGFRvIII-specific recombinant rabbit monoclonal antibody and a PE-conjugated anti-rabbit IgG (H + L) F(ab’)₂ secondary antibody. Data are presented as mean ± SEM from two independent experiments. D RT-PCR analysis of EGFRvIII mRNA expression. Presence of EGFRvIII mRNA was assessed in HNSCC cell lines artificially overexpressing EGFRvIII by RT-PCR. A representative ethidium bromide-stained agarose gel shows PCR products for EGFR (1037 bp) and EGFRvIII (236 bp). WT cell lines (UM-14C WT and UT-24A WT ), their corresponding EGFR KO derivatives, and the three EGFRvIII-overexpressing cell lines generated using different internal promoters were analyzed

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Artificial overexpression of EGFRvIII at different levels in HNSCC cell lines. A Schematic representation of the lentiviral EGFRvIII expression vector. The internal promoters (iP: hPGK, hEF1α and SFFV) drive expression of the EGFRvIII transgene and via an IRES site also the Geneticin resistance gene nptII (neoR). B - D UM-14C KO and UT-24A KO cells were transduced with LV EGFRvIII expression vectors. EGFRvIII-positive EGFR KO cells were further purified by flow sorting, generating new EGFRvIII-expressing cell lines. B and C Flow cytometric analysis of EGFRvIII surface expression after sorting. UM-14C and UT-24A cells were stained with ( B ) a PE-conjugated EGFR antibody for EGFR and EGFRvIII expression. Left: EGFR expression is shown by histogram. Right: Means ± SEM of MFI values of EGFRvIII-positive EGFR KO UM-14C and UT-24A cells as well as the corresponding UM-14C and UT-24A KO, WT and control cells transduced with an empty vector (data from three independent experiments) are shown. Differences between promoter groups within each cell line were analyzed using one-way repeated-measures ANOVA followed by Tukey’s multiple-comparisons test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); ns, not significant. C EGFRvIII expression was additionally determined using an EGFRvIII-specific recombinant rabbit monoclonal antibody and a PE-conjugated anti-rabbit IgG (H + L) F(ab’)₂ secondary antibody. Data are presented as mean ± SEM from two independent experiments. D RT-PCR analysis of EGFRvIII mRNA expression. Presence of EGFRvIII mRNA was assessed in HNSCC cell lines artificially overexpressing EGFRvIII by RT-PCR. A representative ethidium bromide-stained agarose gel shows PCR products for EGFR (1037 bp) and EGFRvIII (236 bp). WT cell lines (UM-14C WT and UT-24A WT ), their corresponding EGFR KO derivatives, and the three EGFRvIII-overexpressing cell lines generated using different internal promoters were analyzed

Techniques: Over Expression, Expressing, Plasmid Preparation, Transduction, Purification, Analysis, Staining, Control, Recombinant, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Generated

$Design, enrichment, and cytotoxicity of CAR-positive T-cells. A Lentiviral CAR expression vector expressing the blue fluorescent protein TagBFP and a 2nd generation CAR construct. The vector contains either CD19, EGFR, or EGFRvIII A–E -specific scFvs, a CD34-derived hinge region (c6) followed by the CD28 transmembrane (TM) and cytoplasmic (CP) domains and the CD3ζ signaling chain. A BFP reporter gene is co-expressed via a T2A sequence. Transgene expression is driven by the MPSV promoter. B Transduction and enrichment efficiency of CAR-positive T-cells. Transduced T-cells were analyzed by flow cytometry before magnetic separation (Pre-MACS), in the flow-through fraction and after MACS selection (Post-MACS). CAR-positive cells were quantified based on BFP co-expression. Representative histograms show CAR expression (x-axis) plotted against normalized cell count (y-axis). CAR-positive cells are shown as mean ± SEM ( n = 3). C CAR T-cell cytotoxicity. The target cell lines UM-14C KO +EGFRvIII high and DK-MG were co-cultured with CD19, EGFR and EGFRvIII A–E CAR T-cells or untransduced T-cells (no virus) for 16 to 20 h and target cell viability was assessed using a luciferase-based viability assay. Target cell lysis according to the E: T ratio (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from independent healthy donors, with n = 5 for UM-14C KO +EGFRvIII high and n = 3 for DK-MG. Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD19 CAR control$

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Design, enrichment, and cytotoxicity of CAR-positive T-cells. A Lentiviral CAR expression vector expressing the blue fluorescent protein TagBFP and a 2nd generation CAR construct. The vector contains either CD19, EGFR, or EGFRvIII A–E -specific scFvs, a CD34-derived hinge region (c6) followed by the CD28 transmembrane (TM) and cytoplasmic (CP) domains and the CD3ζ signaling chain. A BFP reporter gene is co-expressed via a T2A sequence. Transgene expression is driven by the MPSV promoter. B Transduction and enrichment efficiency of CAR-positive T-cells. Transduced T-cells were analyzed by flow cytometry before magnetic separation (Pre-MACS), in the flow-through fraction and after MACS selection (Post-MACS). CAR-positive cells were quantified based on BFP co-expression. Representative histograms show CAR expression (x-axis) plotted against normalized cell count (y-axis). CAR-positive cells are shown as mean ± SEM ( n = 3). C CAR T-cell cytotoxicity. The target cell lines UM-14C KO +EGFRvIII high and DK-MG were co-cultured with CD19, EGFR and EGFRvIII A–E CAR T-cells or untransduced T-cells (no virus) for 16 to 20 h and target cell viability was assessed using a luciferase-based viability assay. Target cell lysis according to the E: T ratio (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from independent healthy donors, with n = 5 for UM-14C KO +EGFRvIII high and n = 3 for DK-MG. Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD19 CAR control

Techniques: Expressing, Plasmid Preparation, Construct, Derivative Assay, Sequencing, Transduction, Flow Cytometry, Selection, Cell Characterization, Cell Culture, Virus, Luciferase, Viability Assay, Lysis, Analysis, Control

CAR T-cell cytotoxicity against different HNSCC cell lines. Different WT and genetically engineered UM-14C/UT-24A cells were co-cultured with CD19, EGFR, EGFRvIII D or EGFRvIII E CAR T-cells or untransduced (no virus) T-cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD19 CAR control

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: CAR T-cell cytotoxicity against different HNSCC cell lines. Different WT and genetically engineered UM-14C/UT-24A cells were co-cultured with CD19, EGFR, EGFRvIII D or EGFRvIII E CAR T-cells or untransduced (no virus) T-cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD19 CAR control

Techniques: Cell Culture, Virus, Cytotoxicity Assay, Lysis, Analysis, Control

Concept, design and cytotoxicity of CAR and nsCAR T-cells. A Schematic representation of CAR/nsCAR co-expression strategies. 2nd generation CARs were combined with non-signaling CARs, which lack signaling domains and provide additional antigen/epitope engagement. B Lentiviral expression vectors encoding CARs or nsCARs. The 2nd generation CAR vector contains either CD44v6, CD33, or EGFRvIII D−E -specific scFvs, the c6-hinge, CD28 transmembrane and cytoplasmic regions, and the CD3ζ chain. An mCherry reporter was co-expressed via a T2A sequence. The nsCAR contains EGFR- or CD19-specific scFvs, the n3-hinge, and the CD8 transmembrane domain. C Killing efficiency of CAR and nsCAR T-cells against WT and genetically engineered UM-14C cells. Primary human T-cells were singly transduced with lentiviral constructs encoding CAR or nsCARs and CAR- or nsCAR-positive T-cells (> 90%) were co-cultured with UM-14C cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD33 CAR control

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Concept, design and cytotoxicity of CAR and nsCAR T-cells. A Schematic representation of CAR/nsCAR co-expression strategies. 2nd generation CARs were combined with non-signaling CARs, which lack signaling domains and provide additional antigen/epitope engagement. B Lentiviral expression vectors encoding CARs or nsCARs. The 2nd generation CAR vector contains either CD44v6, CD33, or EGFRvIII D−E -specific scFvs, the c6-hinge, CD28 transmembrane and cytoplasmic regions, and the CD3ζ chain. An mCherry reporter was co-expressed via a T2A sequence. The nsCAR contains EGFR- or CD19-specific scFvs, the n3-hinge, and the CD8 transmembrane domain. C Killing efficiency of CAR and nsCAR T-cells against WT and genetically engineered UM-14C cells. Primary human T-cells were singly transduced with lentiviral constructs encoding CAR or nsCARs and CAR- or nsCAR-positive T-cells (> 90%) were co-cultured with UM-14C cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analysis was performed using two-way repeated-measures ANOVA across the tested E: T ratios, followed by Dunnett’s multiple-comparisons test versus the CD33 CAR control

Techniques: Expressing, Plasmid Preparation, Sequencing, Transduction, Construct, Cell Culture, Cytotoxicity Assay, Lysis, Analysis, Control

Killing efficiency of CAR/nsCAR T-cells. Primary human T-cells were singly or doubly transduced, and CAR-positive T-cells, including CAR/nsCAR double-positive cells, were enriched using CD34 microbeads. Cells were co-cultured with UM-14C cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analyses were performed using two-way repeated-measures ANOVA across the tested E: T ratios, with donor as the matched experimental unit. Prespecified comparisons assessed the effect of EGFR nsCAR co-expression relative to the corresponding CD19 nsCAR control condition

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Killing efficiency of CAR/nsCAR T-cells. Primary human T-cells were singly or doubly transduced, and CAR-positive T-cells, including CAR/nsCAR double-positive cells, were enriched using CD34 microbeads. Cells were co-cultured with UM-14C cells for 16 to 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analyses were performed using two-way repeated-measures ANOVA across the tested E: T ratios, with donor as the matched experimental unit. Prespecified comparisons assessed the effect of EGFR nsCAR co-expression relative to the corresponding CD19 nsCAR control condition

Techniques: Cell Culture, Cytotoxicity Assay, Lysis, Expressing, Control

Killing dynamics of CAR/nsCAR T-cells. Primary human T-cells were transduced with an EGFRvIII D/E or CD44v6-specific CAR alone or in combination with additional EGFR- or CD19-specific nsCAR constructs. CAR-positive T-cells, including CAR/nsCAR double-positive cells, were enriched using CD34 microbeads and co-cultured with UM-14C KO +EGFRvIII mid cells for 6, 10, and 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analyses were performed separately for each time point using two-way repeated-measures ANOVA across the tested E: T ratios, with donor as the matched experimental unit. Prespecified comparisons assessed the effect of EGFR nsCAR co-expression relative to the corresponding CD19 nsCAR control condition

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Killing dynamics of CAR/nsCAR T-cells. Primary human T-cells were transduced with an EGFRvIII D/E or CD44v6-specific CAR alone or in combination with additional EGFR- or CD19-specific nsCAR constructs. CAR-positive T-cells, including CAR/nsCAR double-positive cells, were enriched using CD34 microbeads and co-cultured with UM-14C KO +EGFRvIII mid cells for 6, 10, and 20 h and target cell viability was assessed using an MTS-based cytotoxicity assay. Target cell lysis at different E: T ratios (x-axis) is plotted against the percentage of lysed target cells (y-axis). Data represent mean ± SEM of three independent biological experiments using T-cells from three independent healthy donors ( n = 3). Statistical analyses were performed separately for each time point using two-way repeated-measures ANOVA across the tested E: T ratios, with donor as the matched experimental unit. Prespecified comparisons assessed the effect of EGFR nsCAR co-expression relative to the corresponding CD19 nsCAR control condition

Techniques: Transduction, Construct, Cell Culture, Cytotoxicity Assay, Lysis, Expressing, Control

Visualization of the IS. The IS was analyzed by fluorescence microscopy, visualizing the EGFRvIII D CAR (FITC-conjugated CD34 antibody, green) and EGFR or CD19 nsCAR (PE-Vio770-conjugated NGFR antibody, red) proteins on MACS-enriched CAR/nsCAR T-cells (BFP expression, blue) in relation to UM-14C KO +EGFRvIII high cells (phase contrast). Contrast settings (channels: PH, 377/447 nm, 470/525 nm and 545/620 nm) were adjusted to optimize IS visualization

Journal: BMC Cancer

Article Title: Co-expression of non-signaling CARs enhances EGFRvIII-specific CAR T-cell cytotoxicity against head and neck squamous cell carcinoma

doi: 10.1186/s12885-026-17058-z

Figure Lengend Snippet: Visualization of the IS. The IS was analyzed by fluorescence microscopy, visualizing the EGFRvIII D CAR (FITC-conjugated CD34 antibody, green) and EGFR or CD19 nsCAR (PE-Vio770-conjugated NGFR antibody, red) proteins on MACS-enriched CAR/nsCAR T-cells (BFP expression, blue) in relation to UM-14C KO +EGFRvIII high cells (phase contrast). Contrast settings (channels: PH, 377/447 nm, 470/525 nm and 545/620 nm) were adjusted to optimize IS visualization

Techniques: Fluorescence, Microscopy, Expressing, Phase Contrast

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