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Jackson Immuno flow cytometric analysis
Electroporation of mRNA encoding anti-EpCAM CAR in human T cells and cytotoxicity of the CAR-modified T cells ( A ) Schematic diagrams of mRNA CAR constructs used in this study. ( B ) Flow <t>cytometric</t> analysis to examine anti-EpCAM CAR expression on T cells after electroporation. The cells were collected 24 hours after electroporation for analysis. ( C ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in T cells. The cells were collected 24 hours after electroporation for analysis. The endogenous CD3ζ was stained as an internal loading control. ( D ) In vitro cell lysis. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM RNA CAR-modified T cells against EpCAM-positive human cancer cell lines HRT-18G and SW620, as well as EpCAM-negative ovarian cancer cell line PA-1. mGFP RNA CAR-modified T cells were included as a control. Mean ± SD of three validation runs is represented. *** p
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1) Product Images from "Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis"

Article Title: Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis

Journal: Oncotarget

doi: 10.18632/oncotarget.14592

Electroporation of mRNA encoding anti-EpCAM CAR in human T cells and cytotoxicity of the CAR-modified T cells ( A ) Schematic diagrams of mRNA CAR constructs used in this study. ( B ) Flow cytometric analysis to examine anti-EpCAM CAR expression on T cells after electroporation. The cells were collected 24 hours after electroporation for analysis. ( C ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in T cells. The cells were collected 24 hours after electroporation for analysis. The endogenous CD3ζ was stained as an internal loading control. ( D ) In vitro cell lysis. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM RNA CAR-modified T cells against EpCAM-positive human cancer cell lines HRT-18G and SW620, as well as EpCAM-negative ovarian cancer cell line PA-1. mGFP RNA CAR-modified T cells were included as a control. Mean ± SD of three validation runs is represented. *** p
Figure Legend Snippet: Electroporation of mRNA encoding anti-EpCAM CAR in human T cells and cytotoxicity of the CAR-modified T cells ( A ) Schematic diagrams of mRNA CAR constructs used in this study. ( B ) Flow cytometric analysis to examine anti-EpCAM CAR expression on T cells after electroporation. The cells were collected 24 hours after electroporation for analysis. ( C ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in T cells. The cells were collected 24 hours after electroporation for analysis. The endogenous CD3ζ was stained as an internal loading control. ( D ) In vitro cell lysis. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM RNA CAR-modified T cells against EpCAM-positive human cancer cell lines HRT-18G and SW620, as well as EpCAM-negative ovarian cancer cell line PA-1. mGFP RNA CAR-modified T cells were included as a control. Mean ± SD of three validation runs is represented. *** p

Techniques Used: Electroporation, Modification, Construct, Flow Cytometry, Expressing, Western Blot, Staining, In Vitro, Lysis, Cytotoxicity Assay

Generation and expansion of EpCAM-specific CART cells ( A ) Schematics of lentiviral vectors used in the study. The scFv region that recognizes EpCAM was derived from 4D5MOC-B humanized mAb. Anti-EpCAM CAR contains the CD28 and 4-1BB co-stimulatory domains and the CD3zeta T cell activation domain. mGFP CAR control vector was constructed using the GFP sequence instead of EpCAM-specific scFv. ( B ) Schematic illustration of EpCAM-specific CART cell generation and expansion. ( C ) Flow cytometric analysis to detect the surface expression of anti-EpCAM CAR on the genetically modified T cells before and after 21 days of co-culturing with K562A-EpCAM aAPCs using anti-mouse IgG Fab and anti-human CD3 antibodies. Representative FACS plots are shown. ( D ) Increase in the number of CD3+CAR+ T cells over the co-culturing with K562A-EpCAM cells. The number of initially seeded T cells was 2E4 per well. Results from five different PBMC samples are shown. ( E ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in modified T cells 21 days after co-culture with K562A-EpCAM cells. The endogenous CD3ζ was stained as an internal loading control. Lane 1: unmodified T cells; Lane 2: T cells transduced with mGFP CAR lentiviral vectors; Lane 3: T cells transduced with anti-EpCAM CAR lentiviral vectors. ( F ) Phenotyping of the propagated EpCAM-specific CART cells. T cells collected after 21 days of co-culture with K562A-EpCAM cells were assessed by flow cytometry. T cells were gated for the presence of naive (T N , CCR7+CD45RA+), central memory (T CM , CCR7+CD45RA-), effector memory (T EM , CCR7-CD45RA-), and terminally differentiated effector T cells (T EFF , CCR7-CD45RA+). Results from five different PBMC samples are shown.
Figure Legend Snippet: Generation and expansion of EpCAM-specific CART cells ( A ) Schematics of lentiviral vectors used in the study. The scFv region that recognizes EpCAM was derived from 4D5MOC-B humanized mAb. Anti-EpCAM CAR contains the CD28 and 4-1BB co-stimulatory domains and the CD3zeta T cell activation domain. mGFP CAR control vector was constructed using the GFP sequence instead of EpCAM-specific scFv. ( B ) Schematic illustration of EpCAM-specific CART cell generation and expansion. ( C ) Flow cytometric analysis to detect the surface expression of anti-EpCAM CAR on the genetically modified T cells before and after 21 days of co-culturing with K562A-EpCAM aAPCs using anti-mouse IgG Fab and anti-human CD3 antibodies. Representative FACS plots are shown. ( D ) Increase in the number of CD3+CAR+ T cells over the co-culturing with K562A-EpCAM cells. The number of initially seeded T cells was 2E4 per well. Results from five different PBMC samples are shown. ( E ) Western blot analysis using a CD3ζ-specific antibody confirms the CAR expression in modified T cells 21 days after co-culture with K562A-EpCAM cells. The endogenous CD3ζ was stained as an internal loading control. Lane 1: unmodified T cells; Lane 2: T cells transduced with mGFP CAR lentiviral vectors; Lane 3: T cells transduced with anti-EpCAM CAR lentiviral vectors. ( F ) Phenotyping of the propagated EpCAM-specific CART cells. T cells collected after 21 days of co-culture with K562A-EpCAM cells were assessed by flow cytometry. T cells were gated for the presence of naive (T N , CCR7+CD45RA+), central memory (T CM , CCR7+CD45RA-), effector memory (T EM , CCR7-CD45RA-), and terminally differentiated effector T cells (T EFF , CCR7-CD45RA+). Results from five different PBMC samples are shown.

Techniques Used: Derivative Assay, Activation Assay, Plasmid Preparation, Construct, Sequencing, Flow Cytometry, Expressing, Genetically Modified, FACS, Western Blot, Modification, Co-Culture Assay, Staining, Transduction, Cytometry

In vitro cell lysis of EpCAM-positive tumour cells with T cells genetically modified by a lentiviral anti-EpCAM CAR vector ( A ) EpCAM expression on ovarian cancer cells as demonstrated by flow cytometric analysis. Three EpCAM-positive human epithelial ovarian cancer cell lines (CAOV3, SW626, and SKOV3-luc) and one EpCAM-negative human ovarian cancer cell line (PA-1) were analysed. ( B ) % cytotoxicity. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM CAR-expressing T cells against EpCAM-positive ovarian cancer cell lines. Specific cell lysis was demonstrated by including EpCAM-negative PA-1 cells and the use of mGFP CAR. Mean ± SD of three validation runs is represented.
Figure Legend Snippet: In vitro cell lysis of EpCAM-positive tumour cells with T cells genetically modified by a lentiviral anti-EpCAM CAR vector ( A ) EpCAM expression on ovarian cancer cells as demonstrated by flow cytometric analysis. Three EpCAM-positive human epithelial ovarian cancer cell lines (CAOV3, SW626, and SKOV3-luc) and one EpCAM-negative human ovarian cancer cell line (PA-1) were analysed. ( B ) % cytotoxicity. Delfia EuTDA cytotoxicity assay (3 hours EuTDA culturing) was used to assess the cytotoxicity of anti-EpCAM CAR-expressing T cells against EpCAM-positive ovarian cancer cell lines. Specific cell lysis was demonstrated by including EpCAM-negative PA-1 cells and the use of mGFP CAR. Mean ± SD of three validation runs is represented.

Techniques Used: In Vitro, Lysis, Genetically Modified, Plasmid Preparation, Expressing, Flow Cytometry, Cytotoxicity Assay

Preparation of a K562 aAPC line for expansion of anti-EpCAM CAR-expressing T cells ( A ) Schematic diagrams of plasmid vectors used for the generation of the aAPC cell line. The costimulatory molecule vector (top) was used for transfection of wild-type K562 cells to generate puromycin-resistant K562 cells expressing CD64, CD137L and CD86 (K562A). K562A cells were further modified by co-transfection with the ZFN vector and EpCAM DNA donor vector (bottom) for AAVS1 locus-specific gene insertion to generate puromycin- and neomycin-resistant aAPCs expressing EpCAM (K562A-EpCAM). ( B ) PCR genome typing to demonstrate the AAVS1 locus-specific gene insertion of the EpCAM gene, as indicated by the presence of one single 1.5-kb band. ( C ) Phenotype analysis of K562A-EpCAM cells. Flow cytometric analysis demonstrates the surface expression of CD64, CD86, CD137L, and EpCAM. In the panels for CD64, CD86, and CD137L, left curves: isotype controls; right curves: antibodies. In the panel for EpCAM, left curve: K562 parental cells; right curve: K562A-EpCAM cells.
Figure Legend Snippet: Preparation of a K562 aAPC line for expansion of anti-EpCAM CAR-expressing T cells ( A ) Schematic diagrams of plasmid vectors used for the generation of the aAPC cell line. The costimulatory molecule vector (top) was used for transfection of wild-type K562 cells to generate puromycin-resistant K562 cells expressing CD64, CD137L and CD86 (K562A). K562A cells were further modified by co-transfection with the ZFN vector and EpCAM DNA donor vector (bottom) for AAVS1 locus-specific gene insertion to generate puromycin- and neomycin-resistant aAPCs expressing EpCAM (K562A-EpCAM). ( B ) PCR genome typing to demonstrate the AAVS1 locus-specific gene insertion of the EpCAM gene, as indicated by the presence of one single 1.5-kb band. ( C ) Phenotype analysis of K562A-EpCAM cells. Flow cytometric analysis demonstrates the surface expression of CD64, CD86, CD137L, and EpCAM. In the panels for CD64, CD86, and CD137L, left curves: isotype controls; right curves: antibodies. In the panel for EpCAM, left curve: K562 parental cells; right curve: K562A-EpCAM cells.

Techniques Used: Expressing, Plasmid Preparation, Transfection, Modification, Cotransfection, Polymerase Chain Reaction, Flow Cytometry

2) Product Images from "Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis"

Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109352

Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
Figure Legend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.
Figure Legend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

Techniques Used: Immunofluorescence, Staining, Cell Culture

3) Product Images from "Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus ▿"

Article Title: Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.05163-11

Immunolocalization of HEA. Immunolocalization of HEA was determined by Alexa Fluor 594-labeled goat anti-mouse secondary antibodies (red), and nuclear staining was identified by DAPI (blue). MAb 9B4 acted as an IgG1 isotype control antibody in this study.
Figure Legend Snippet: Immunolocalization of HEA. Immunolocalization of HEA was determined by Alexa Fluor 594-labeled goat anti-mouse secondary antibodies (red), and nuclear staining was identified by DAPI (blue). MAb 9B4 acted as an IgG1 isotype control antibody in this study.

Techniques Used: Labeling, Staining

4) Product Images from "Cross-comparison of Protein Recognition of Sialic Acid Diversity on Two Novel Sialoglycan Microarrays *"

Article Title: Cross-comparison of Protein Recognition of Sialic Acid Diversity on Two Novel Sialoglycan Microarrays *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.359323

Selective recognition of sialoglycans by plant lectins. Biotinylated lectins were assayed using the protocol optimized for Array 1 (as detailed under “Experimental Procedures”) and detected with 1.5 μg/ml Cy3-streptavidin. a , selective
Figure Legend Snippet: Selective recognition of sialoglycans by plant lectins. Biotinylated lectins were assayed using the protocol optimized for Array 1 (as detailed under “Experimental Procedures”) and detected with 1.5 μg/ml Cy3-streptavidin. a , selective

Techniques Used:

5) Product Images from "α-ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1-dependent adrenal activation"

Article Title: α-ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1-dependent adrenal activation

Journal: bioRxiv

doi: 10.1101/796037

Adrenal specific overexpression of OXGR1 enhances stimulatory effects of AKG on thermogenesis and lipolysis (A). The validation of OXGR1 overexpression. The mRNA expression of OXGR1 was determined in the adrenal glands from male WT control, WT injected with HBAAV2/9-GFP, and WT injected with HBAAV2/9-OXGR1 (OXGR1OE AG ) mice (n=5 per group). (B-C). Body weight gain (B) and cumulative food intake (C) of OXGR1OE AG . Male C57BL/6 mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP or HBAAV2/9-OXGR1. Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group). (D-E). Representative image of body composition (D) and fat and lean mass index (E) of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 8 per group). (F-G). Weight index of gWAT (F) and iWAT (G) in male OXGR1OE AG mice treated with AKG for 12 weeks (n = 6 per group). (H-I). Immunoblots (H) and quantification (I) of p-HSL and ATGL protein in the gWAT of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 4 per group). (J). Immunoblots and quantification of UCP1 protein in the BAT of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 4 per group). (K) Serum E level in male OXGR1OE AG mice treated with AKG for 12 weeks (n= 8 per group). (L-O). Oxygen consumption (L-M) and RER (N-O) of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 8 per group). (P-Q). Representative images (P) and quantification (Q) of gWAT and iWAT HE staining from male OXGR1OE AG mice treated with AKG for 12 weeks (n = 6 per group). (R-S). Representative images (R) and quantification (S) of p-HSL DAB staining from male OXGR1OE AG mice treated with AKG for 12 weeks (n = 6 per group). Results are presented as mean ± SEM. In (A), ** p≤0.01 by non-paired Student’s t test. In (B-C), *p≤0.05, **p≤0.01 by two-way ANOVA followed by post hoc Bonferroni tests. In (E-G), (I-K), (M), (O), (Q) and (S), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Turkey’s tests.
Figure Legend Snippet: Adrenal specific overexpression of OXGR1 enhances stimulatory effects of AKG on thermogenesis and lipolysis (A). The validation of OXGR1 overexpression. The mRNA expression of OXGR1 was determined in the adrenal glands from male WT control, WT injected with HBAAV2/9-GFP, and WT injected with HBAAV2/9-OXGR1 (OXGR1OE AG ) mice (n=5 per group). (B-C). Body weight gain (B) and cumulative food intake (C) of OXGR1OE AG . Male C57BL/6 mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP or HBAAV2/9-OXGR1. Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group). (D-E). Representative image of body composition (D) and fat and lean mass index (E) of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 8 per group). (F-G). Weight index of gWAT (F) and iWAT (G) in male OXGR1OE AG mice treated with AKG for 12 weeks (n = 6 per group). (H-I). Immunoblots (H) and quantification (I) of p-HSL and ATGL protein in the gWAT of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 4 per group). (J). Immunoblots and quantification of UCP1 protein in the BAT of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 4 per group). (K) Serum E level in male OXGR1OE AG mice treated with AKG for 12 weeks (n= 8 per group). (L-O). Oxygen consumption (L-M) and RER (N-O) of male OXGR1OE AG mice treated with AKG for 12 weeks (n = 8 per group). (P-Q). Representative images (P) and quantification (Q) of gWAT and iWAT HE staining from male OXGR1OE AG mice treated with AKG for 12 weeks (n = 6 per group). (R-S). Representative images (R) and quantification (S) of p-HSL DAB staining from male OXGR1OE AG mice treated with AKG for 12 weeks (n = 6 per group). Results are presented as mean ± SEM. In (A), ** p≤0.01 by non-paired Student’s t test. In (B-C), *p≤0.05, **p≤0.01 by two-way ANOVA followed by post hoc Bonferroni tests. In (E-G), (I-K), (M), (O), (Q) and (S), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Turkey’s tests.

Techniques Used: Over Expression, Expressing, Injection, Mouse Assay, Western Blot, Staining

OXGR1 is required for metabolic beneficial effects of resistance exercise (A). Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups, receiving non-exercise or resistance exercise for 14 days. (n = 8 per group). (B). Exercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of non-exercise control group for each genotype (n = 8 per group). (C). Exercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice were subtracted by the average fat mass of non-exercise control group for each genotype (n = 8 per group). (D). Cumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group). (E-F). Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group). (G). Body composition of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group). (H). Serum AKG levels of male OXGR1KO mice after resistance exercise. Male OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise. (I). Serum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group). (J-K). The mRNA expression of UCP1 (J) in the BAT or HSL and ATGL (K) in the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group). (L-O). Oxygen consumption (L-M) and RER (N-O) in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group). Results are presented as mean ± SEM. In (A-D) *p≤0.05, ** p≤0.01 by two-way ANOVA followed by post hoc Bonferroni tests. In (H), *p≤0.05 by non-paired Student’s t test. In (E-G), (I-K), (M) and (O), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey’s tests.
Figure Legend Snippet: OXGR1 is required for metabolic beneficial effects of resistance exercise (A). Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups, receiving non-exercise or resistance exercise for 14 days. (n = 8 per group). (B). Exercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of non-exercise control group for each genotype (n = 8 per group). (C). Exercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice were subtracted by the average fat mass of non-exercise control group for each genotype (n = 8 per group). (D). Cumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group). (E-F). Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group). (G). Body composition of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group). (H). Serum AKG levels of male OXGR1KO mice after resistance exercise. Male OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise. (I). Serum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group). (J-K). The mRNA expression of UCP1 (J) in the BAT or HSL and ATGL (K) in the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group). (L-O). Oxygen consumption (L-M) and RER (N-O) in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group). Results are presented as mean ± SEM. In (A-D) *p≤0.05, ** p≤0.01 by two-way ANOVA followed by post hoc Bonferroni tests. In (H), *p≤0.05 by non-paired Student’s t test. In (E-G), (I-K), (M) and (O), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey’s tests.

Techniques Used: Mouse Assay, Expressing

Acute in vivo effects of AKG (A-B). Serum levels of NE (A) and NEFA (B) in male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 5-6 per group). (C). The mRNA expression of thermogenic genes in male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 5-6 per group). (D). Immunoblots and quantification of UCP1 in BAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 3 per group). (E). Immunoblots and quantification of PLCβ and pErk in the adrenal glands of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 3 per group). (F-I). Physical activity (pedometer, F-G) and heart rate (H-I) of male mice i.p. injected with 10 mg/kg AKG or saline at 7:00 am (n = 8 per group). (J-L). Blood pressure of male mice i.p. inject ed with 10 mg/kg AKG or saline (n = 8 per group). Results are presented as mean ± SEM. In (A-E), (G), (I), (L), *p≤0.05, **p≤0.01, ***p≤0.01 by non-paired Student’s t-test. In (F), (H) and (J-K), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests.
Figure Legend Snippet: Acute in vivo effects of AKG (A-B). Serum levels of NE (A) and NEFA (B) in male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 5-6 per group). (C). The mRNA expression of thermogenic genes in male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 5-6 per group). (D). Immunoblots and quantification of UCP1 in BAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 3 per group). (E). Immunoblots and quantification of PLCβ and pErk in the adrenal glands of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of saline or AKG (10 mg/kg) (n = 3 per group). (F-I). Physical activity (pedometer, F-G) and heart rate (H-I) of male mice i.p. injected with 10 mg/kg AKG or saline at 7:00 am (n = 8 per group). (J-L). Blood pressure of male mice i.p. inject ed with 10 mg/kg AKG or saline (n = 8 per group). Results are presented as mean ± SEM. In (A-E), (G), (I), (L), *p≤0.05, **p≤0.01, ***p≤0.01 by non-paired Student’s t-test. In (F), (H) and (J-K), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests.

Techniques Used: In Vivo, Mouse Assay, Injection, Expressing, Western Blot, Activity Assay

AKG increases fat thermogenesis and lipolysis (A-D). Oxygen consumption (A-B) and respiratory exchange ratio (RER, C-D) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group). (E). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group). (F-G). Representative images (F) and quantification (G) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group). (H). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group). (I-J). Immunoblots (I) and quantification (J) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group). (K-L). DAB staining (K) and quantification (L) of p-HSL in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group). (M–O). The mRNA expression of thermogenic genes (M) and DAB staining (N) and quantification (O) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 6-8 per group). (P). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group). (Q–U). Serum levels of NEFA (Q), E (R), NE (S), T3 (T), and T4 (U) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group). Results are presented as mean ± SEM. In (B), (D-E), (G-H), (J), (L-M), and (O-U), *p≤0.05, **p≤0.01, and ***p≤0.001 by non-paired Student’s t test.
Figure Legend Snippet: AKG increases fat thermogenesis and lipolysis (A-D). Oxygen consumption (A-B) and respiratory exchange ratio (RER, C-D) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group). (E). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group). (F-G). Representative images (F) and quantification (G) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group). (H). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group). (I-J). Immunoblots (I) and quantification (J) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group). (K-L). DAB staining (K) and quantification (L) of p-HSL in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group). (M–O). The mRNA expression of thermogenic genes (M) and DAB staining (N) and quantification (O) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 6-8 per group). (P). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group). (Q–U). Serum levels of NEFA (Q), E (R), NE (S), T3 (T), and T4 (U) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group). Results are presented as mean ± SEM. In (B), (D-E), (G-H), (J), (L-M), and (O-U), *p≤0.05, **p≤0.01, and ***p≤0.001 by non-paired Student’s t test.

Techniques Used: Mouse Assay, Expressing, Western Blot, Staining

Adrenal specific reexpression of OXGR1 rescues the stimulatory effects of AKG on thermogenesis and lipolysis (A). Serum E level in male OXGR1KO mice. At 12 weeks of age, male control or OXGR1KO mice were switched to HFD and received tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group). (B). Immunoblots and quantification of UCP1 protein expression in the BAT of male OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group). (C-D). Representative images (C) and quantification (D) of iWAT and gWAT HE staining from male OXGR1KO mice treated with AKG for 13 weeks (n = 6 per group). (E-F). Representative images (E) and quantification (F) of p-HSL DAB staining from male OXGR1KO mice treated with AKG for 13 weeks (n = 6 per group). (G). The validation of OXGR1 reexpression. The mRNA expression of OXGR1 was determined in the adrenal glands from male WT control, OXGR1KO injected with HBAAV2/9-GFP, and OXGR1KO injected with HBAAV2/9-OXGR1 (OXGR1RE AG ) mice. (H). Serum E level in male OXGR1RE AG . Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP or HBAAV2/9-OXGR1. Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or 2% AKG for 13 weeks. (n = 6 per group). (I). Immunoblots and quantification of UCP1 protein expression in the BAT of OXGR1RE AG mice treated with AKG for 13 weeks (n = 4 per group). (J-K). Representative images (J) and quantification (K) of iWAT and gWAT HE staining from OXGR1RE AG mice treated with AKG for 13 weeks (n = 6 per group). (L-M). Representative images (L) and quantification (M) of p-HSL DAB staining from OXGR1RE AG mice treated with AKG for 13 weeks (n = 6 per group). Results are presented as mean ± SEM. In (A-B), (D), (F), (H-I), (K) and (M), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Turkey’s tests.
Figure Legend Snippet: Adrenal specific reexpression of OXGR1 rescues the stimulatory effects of AKG on thermogenesis and lipolysis (A). Serum E level in male OXGR1KO mice. At 12 weeks of age, male control or OXGR1KO mice were switched to HFD and received tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group). (B). Immunoblots and quantification of UCP1 protein expression in the BAT of male OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group). (C-D). Representative images (C) and quantification (D) of iWAT and gWAT HE staining from male OXGR1KO mice treated with AKG for 13 weeks (n = 6 per group). (E-F). Representative images (E) and quantification (F) of p-HSL DAB staining from male OXGR1KO mice treated with AKG for 13 weeks (n = 6 per group). (G). The validation of OXGR1 reexpression. The mRNA expression of OXGR1 was determined in the adrenal glands from male WT control, OXGR1KO injected with HBAAV2/9-GFP, and OXGR1KO injected with HBAAV2/9-OXGR1 (OXGR1RE AG ) mice. (H). Serum E level in male OXGR1RE AG . Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP or HBAAV2/9-OXGR1. Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or 2% AKG for 13 weeks. (n = 6 per group). (I). Immunoblots and quantification of UCP1 protein expression in the BAT of OXGR1RE AG mice treated with AKG for 13 weeks (n = 4 per group). (J-K). Representative images (J) and quantification (K) of iWAT and gWAT HE staining from OXGR1RE AG mice treated with AKG for 13 weeks (n = 6 per group). (L-M). Representative images (L) and quantification (M) of p-HSL DAB staining from OXGR1RE AG mice treated with AKG for 13 weeks (n = 6 per group). Results are presented as mean ± SEM. In (A-B), (D), (F), (H-I), (K) and (M), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Turkey’s tests.

Techniques Used: Mouse Assay, Western Blot, Expressing, Staining, Injection

Metabolic effects of AKG is mediated by adrenergic stimulation of thermogenesis and lipolysis (A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group). (B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hr cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group). (D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group). (E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group). (F-I). Oxygen consumption (F-G) and RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day. (J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) and serum NEFA (N) of shame or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group). (O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6h cold exposure at 4℃ in shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group). (Q). The mRNA expression of themogenic genes in the BAT of shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group). (R-S) Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group). (T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group). Results are presented as mean ± SEM. In (A), *p≤0.05 by non-paired Student’s t test compared with before injection. In (C-E), *p≤0.05, **p≤ 0.01 by non-paired Student’s t test. In (J-K), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests. In (G), (I), (L-N), (P-Q), (S) and (U), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey’s tests.
Figure Legend Snippet: Metabolic effects of AKG is mediated by adrenergic stimulation of thermogenesis and lipolysis (A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group). (B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hr cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group). (D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group). (E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group). (F-I). Oxygen consumption (F-G) and RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day. (J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) and serum NEFA (N) of shame or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group). (O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6h cold exposure at 4℃ in shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group). (Q). The mRNA expression of themogenic genes in the BAT of shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group). (R-S) Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group). (T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of shame or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group). Results are presented as mean ± SEM. In (A), *p≤0.05 by non-paired Student’s t test compared with before injection. In (C-E), *p≤0.05, **p≤ 0.01 by non-paired Student’s t test. In (J-K), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests. In (G), (I), (L-N), (P-Q), (S) and (U), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey’s tests.

Techniques Used: Concentration Assay, Mouse Assay, Injection, Western Blot, Expressing

Metabolic effects of AKG in mice fed on chow. (A-B). Body weight gain (A) and cumulative food intake (B) of male C57BL/6 mice. At 12 weeks of age, chow-fed male mice were divided into two groups, receiving tap water or water supplemented with 2% AKG for 6 weeks (n = 8 per group). (C-D). Body composition (C) and tissue weight (D) of male mice treated with AKG for 6 weeks (n = 7-8 per group). (E-F). Representative images (E) and quantification (F) of gWAT HE staining from male mice treated with AKG for 6 weeks (n = 8 per group). (G-H). Body weight gain (G) and cumulative food intake (H) of female C57BL/6 mice. At 12 weeks of age, chow-fed female mice were divided into two groups, receiving tap water or water supplemented with 2% AKG for 11 weeks (n = 8 per group). (I-J). Body composition (C) and tissue weight (D) of female mice treated with AKG for 11 weeks (n = 8 per group). (K-L). Representative images (K) and quantification (L) of gWAT HE staining from female mice treated with AKG for 11 weeks (n = 8 per group). (M). The mRNA expression of thermogenic genes in BAT of male C57BL/6 mice supplemented with AKG for 6 weeks (n = 6 per group). (N-P). Immunoblots and quantification of UCP1 (N) and representative images of DAB staining (O) and quantification (P) of UCP1 in BAT of male mice supplemented with AKG for 6 weeks (n = 3-6 per group). (Q-U). Serum levels of NEFA (Q), E (R), NE (S), T3 (T), and T4 (U) in male mice supplemented with AKG for 6 weeks (n = 6 per group). Results are presented as mean ± SEM. In (A-B) and (G-H), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests. In (C-D), (F), (I-J), (L-N) and (P-U), *p≤0.05, **p≤0.01, ***p≤0.01 by non-paired Student’s t-test.
Figure Legend Snippet: Metabolic effects of AKG in mice fed on chow. (A-B). Body weight gain (A) and cumulative food intake (B) of male C57BL/6 mice. At 12 weeks of age, chow-fed male mice were divided into two groups, receiving tap water or water supplemented with 2% AKG for 6 weeks (n = 8 per group). (C-D). Body composition (C) and tissue weight (D) of male mice treated with AKG for 6 weeks (n = 7-8 per group). (E-F). Representative images (E) and quantification (F) of gWAT HE staining from male mice treated with AKG for 6 weeks (n = 8 per group). (G-H). Body weight gain (G) and cumulative food intake (H) of female C57BL/6 mice. At 12 weeks of age, chow-fed female mice were divided into two groups, receiving tap water or water supplemented with 2% AKG for 11 weeks (n = 8 per group). (I-J). Body composition (C) and tissue weight (D) of female mice treated with AKG for 11 weeks (n = 8 per group). (K-L). Representative images (K) and quantification (L) of gWAT HE staining from female mice treated with AKG for 11 weeks (n = 8 per group). (M). The mRNA expression of thermogenic genes in BAT of male C57BL/6 mice supplemented with AKG for 6 weeks (n = 6 per group). (N-P). Immunoblots and quantification of UCP1 (N) and representative images of DAB staining (O) and quantification (P) of UCP1 in BAT of male mice supplemented with AKG for 6 weeks (n = 3-6 per group). (Q-U). Serum levels of NEFA (Q), E (R), NE (S), T3 (T), and T4 (U) in male mice supplemented with AKG for 6 weeks (n = 6 per group). Results are presented as mean ± SEM. In (A-B) and (G-H), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests. In (C-D), (F), (I-J), (L-N) and (P-U), *p≤0.05, **p≤0.01, ***p≤0.01 by non-paired Student’s t-test.

Techniques Used: Mouse Assay, Staining, Expressing, Western Blot

6) Product Images from "Base-edited CAR T Cells for combinational therapy against T cell malignancies"

Article Title: Base-edited CAR T Cells for combinational therapy against T cell malignancies

Journal: bioRxiv

doi: 10.1101/2020.07.30.228429

3CAR and 7CAR primary T cells evade fratricide during production. A) Phenotypic analysis of surface antigen CD3 and CD7 expression (top panel) and CAR expression (bottom panel) of CD3/CD28 activated peripheral blood mononuclear cells elcetroporated with sgRNA targeting TRBC and CD7 alongside coBE3 mRNA and subsequently transduced with 3CAR or 7CAR lentiviral vectors at MOI 5. Reduced TCR/CD3 and CD7 expression in edited groups and high level CAR expression with fratricide evasion (dotted red outline) was exhibited (n=4). Self-enrichment effects followed co-culture of 3CAR and 7CAR products (n=2) resulted in enriched TCR - CD7 - 3CAR/7CAR cells (red box). B) Proportion of CD3 or CD7 surface antigen and CAR expression at end of 3CAR (n=4), 7CAR (n=4) production or untransduced (UTD) (n=4) cells. Error bars represent SEM across (n=4) donors. C) Schematic of exonic regions within TRBC and CD7 genes. Red marking in exons 4 of TRBC and CD7 represent genomic translation stop sites followed by 5’ untranslated regions (white boxes). Red triangles with asterisk indicate position of base conversion resulting in premature stop codon formation. D) Representative base EDITR output of Sanger sequencing results from mixed 3CAR/7CAR co-culture DNA PCR amplicons of TRBC and CD7 genomic loci. Sites of intended base conversion highlighted in red boxes. High frequency G- > A (antisense) and C- > T changes within editing window highlighted by red vs blue colour. E) Percentage of G- > A conversions throughout TRBC-targeting protospacer sequence (left), and C- > T conversions throughout CD7-targeting protospacer sequence (right).
Figure Legend Snippet: 3CAR and 7CAR primary T cells evade fratricide during production. A) Phenotypic analysis of surface antigen CD3 and CD7 expression (top panel) and CAR expression (bottom panel) of CD3/CD28 activated peripheral blood mononuclear cells elcetroporated with sgRNA targeting TRBC and CD7 alongside coBE3 mRNA and subsequently transduced with 3CAR or 7CAR lentiviral vectors at MOI 5. Reduced TCR/CD3 and CD7 expression in edited groups and high level CAR expression with fratricide evasion (dotted red outline) was exhibited (n=4). Self-enrichment effects followed co-culture of 3CAR and 7CAR products (n=2) resulted in enriched TCR - CD7 - 3CAR/7CAR cells (red box). B) Proportion of CD3 or CD7 surface antigen and CAR expression at end of 3CAR (n=4), 7CAR (n=4) production or untransduced (UTD) (n=4) cells. Error bars represent SEM across (n=4) donors. C) Schematic of exonic regions within TRBC and CD7 genes. Red marking in exons 4 of TRBC and CD7 represent genomic translation stop sites followed by 5’ untranslated regions (white boxes). Red triangles with asterisk indicate position of base conversion resulting in premature stop codon formation. D) Representative base EDITR output of Sanger sequencing results from mixed 3CAR/7CAR co-culture DNA PCR amplicons of TRBC and CD7 genomic loci. Sites of intended base conversion highlighted in red boxes. High frequency G- > A (antisense) and C- > T changes within editing window highlighted by red vs blue colour. E) Percentage of G- > A conversions throughout TRBC-targeting protospacer sequence (left), and C- > T conversions throughout CD7-targeting protospacer sequence (right).

Techniques Used: Expressing, Transduction, Co-Culture Assay, Sequencing, Polymerase Chain Reaction

3CAR and 7CAR cells effectively clear T cell malignancy in vivo. A) Experimental timeline of GFP + LUC + Jurkat tumour injection (Day 0) and effector T cell injection (Day 4) in n=27 NOD/SCID/γc −/− (NSG) mice. Bioluminescent imaging (BLI) performed biweekly (Days 3-24). Organ harvest post mortem for flow-based characterisation (Day 24). B) NSG mice were infused with 1 x 10 7 GFP+LUC+ Jurkat T cells modified to express mixed CD3 and/or CD7 surface antigens in groups of (n=5) CD3 - CD7 - , (n=4) CD3 + CD7 - , (n=5) CD3 - CD7 + or (n=5) CD3 - CD7 - and imaged on day 3 prior to infusion of 1 x 10 7 TCR - CD7 - 3CAR/7CAR mixed effectors or untransduced (UTD) cells. Leukaemic progression monitored by serial BLI for 24 days and revealed disease progression in animals receiving untransduced T cells (3CAR - 7CAR - ) and in animals engrafted with antigen negative (CD3 - CD7 - ) leukemia. C) Bioluminescence signal of each animal plotted as Average radiance [photons/sec/cm 2 /sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent SEM. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc ****P
Figure Legend Snippet: 3CAR and 7CAR cells effectively clear T cell malignancy in vivo. A) Experimental timeline of GFP + LUC + Jurkat tumour injection (Day 0) and effector T cell injection (Day 4) in n=27 NOD/SCID/γc −/− (NSG) mice. Bioluminescent imaging (BLI) performed biweekly (Days 3-24). Organ harvest post mortem for flow-based characterisation (Day 24). B) NSG mice were infused with 1 x 10 7 GFP+LUC+ Jurkat T cells modified to express mixed CD3 and/or CD7 surface antigens in groups of (n=5) CD3 - CD7 - , (n=4) CD3 + CD7 - , (n=5) CD3 - CD7 + or (n=5) CD3 - CD7 - and imaged on day 3 prior to infusion of 1 x 10 7 TCR - CD7 - 3CAR/7CAR mixed effectors or untransduced (UTD) cells. Leukaemic progression monitored by serial BLI for 24 days and revealed disease progression in animals receiving untransduced T cells (3CAR - 7CAR - ) and in animals engrafted with antigen negative (CD3 - CD7 - ) leukemia. C) Bioluminescence signal of each animal plotted as Average radiance [photons/sec/cm 2 /sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent SEM. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc ****P

Techniques Used: In Vivo, Injection, Mouse Assay, Imaging, Modification

Multiplexed cytidine deamination reduces frequency of dsDNA break-mediated chromosomal translocations. A) Schematic of TRBC locus within chromosome 7 q-arm and CD7 locus within chromosome 17 q-arm highlighted by red line. Four predicted chromosomal translocations generated following simultaneous dsDNA-mediated cleavage at TRBC and CD7 loci. B) Gel electrophoresis of DNA products from either untransduced (UTD) or mixed 3CAR/7CAR co-cultures edited with spCas9 or coBE3 mRNA following PCR amplification with TRBC Fwd - CD7 Fwd, TRBC Rev – CD7 Fwd, TRBC Rev – CD7 Rev and TRBC Fwd- CD7 Rev primer combinations. Positive bands detected at ∼250bp. Control bands are PCR amplicons from of synthesised fusions. C) Histogram showing percentage of digital droplet PCR (ddPCR)-based quantification of four possible predicted translocations (T1-T4) in DNA from mixed 3CAR/7CAR co- cultures edited with spCas9 or coBE3 mRNA.
Figure Legend Snippet: Multiplexed cytidine deamination reduces frequency of dsDNA break-mediated chromosomal translocations. A) Schematic of TRBC locus within chromosome 7 q-arm and CD7 locus within chromosome 17 q-arm highlighted by red line. Four predicted chromosomal translocations generated following simultaneous dsDNA-mediated cleavage at TRBC and CD7 loci. B) Gel electrophoresis of DNA products from either untransduced (UTD) or mixed 3CAR/7CAR co-cultures edited with spCas9 or coBE3 mRNA following PCR amplification with TRBC Fwd - CD7 Fwd, TRBC Rev – CD7 Fwd, TRBC Rev – CD7 Rev and TRBC Fwd- CD7 Rev primer combinations. Positive bands detected at ∼250bp. Control bands are PCR amplicons from of synthesised fusions. C) Histogram showing percentage of digital droplet PCR (ddPCR)-based quantification of four possible predicted translocations (T1-T4) in DNA from mixed 3CAR/7CAR co- cultures edited with spCas9 or coBE3 mRNA.

Techniques Used: Generated, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification

3CAR/7CAR T cells mediate potent killing of T-ALL cells in vitro . In vitro cytotoxicity of 3CAR and 7CAR cells against T-ALL cell lines and primary T-ALL targets. A) 51 Cr labelled Jurkat T cells modified to express CD3 + CD7 + , CD3 + CD7 - , CD3 - CD7 + or CD3 - CD7 - were co-cultured with either 3CAR (white squares), 7CAR (grey squares), mixed 3CAR/7CAR (black squares) or untransduced (white circles) cells at an increasing ratio of effectors:targets (E:T). Error bars represent SEM of (n=3) technical replicates. B) Cytotoxic activity of 3CAR, 7CAR, mixed 3CAR/7CAR or untransduced primary T cell controls against primary patient T-ALL cells. Representative flow cytometry plots gated on CFSE + live T-ALL tumour cells (top panel). Frequency of surface antigens CD3, CD7 (middle panel) and CD19, CD56, (lower panel) gated on CFSE+ live tumour cells.
Figure Legend Snippet: 3CAR/7CAR T cells mediate potent killing of T-ALL cells in vitro . In vitro cytotoxicity of 3CAR and 7CAR cells against T-ALL cell lines and primary T-ALL targets. A) 51 Cr labelled Jurkat T cells modified to express CD3 + CD7 + , CD3 + CD7 - , CD3 - CD7 + or CD3 - CD7 - were co-cultured with either 3CAR (white squares), 7CAR (grey squares), mixed 3CAR/7CAR (black squares) or untransduced (white circles) cells at an increasing ratio of effectors:targets (E:T). Error bars represent SEM of (n=3) technical replicates. B) Cytotoxic activity of 3CAR, 7CAR, mixed 3CAR/7CAR or untransduced primary T cell controls against primary patient T-ALL cells. Representative flow cytometry plots gated on CFSE + live T-ALL tumour cells (top panel). Frequency of surface antigens CD3, CD7 (middle panel) and CD19, CD56, (lower panel) gated on CFSE+ live tumour cells.

Techniques Used: In Vitro, Modification, Cell Culture, Activity Assay, Flow Cytometry

Generation of ‘T v T’ fratricide resistant CAR T cells. A) Schema of base editing for T cells employing 3 rd generation codon optimised cytidine base deaminase (coBE3) fused to deactivated D10A Cas9 nickase and uracil glycosylase inhibitor (UGI) delivered as mRNA along with TRBC and CD7 single guide RNA (sgRNA). C- > U- > T conversion (G- > A antisense strand) resulting in STOP codon. B) Lentiviral transduction of edited cells from step 1 using 3 rd generation lentiviral vectors. Lentiviral plasmid configuration of CD3ε targeting 2 nd generation chimeric antigen receptor comprising OKT3 vL and vH scFv sequence fused to CD8 transmembrane domain (TM), 41BB co-stimulatory and CD3z activation domains under the control of a hPGK promoter. Lentiviral plasmid configuration of CD7 targeting 2 nd generation CAR comprising 3A1e vL and vH scFv sequence fused to CD8TM-41BB-CD3z under the control of a hPGK. C) coBE3 edited T cells devoid of shared antigens TCR/CD3 and CD7 surface receptors expressing either 3CAR or 7CAR evade fratricide and target T-ALL. BE: base editor; APOBEC: (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like); sgRNA: single guide RNA; PAM: protospacer adjacent motif; LTR: long terminal repeat; CMV: cytomegalovirus promoter; CAR: chimeric antigen receptor; cPPT: central polypurine track; U5: untranslated 5’ region; DU3: delta untranslated 3’ region; hPGK: human phosphoglycerate kinase promoter; vL: variable light chain; vH: variable heavy chain.
Figure Legend Snippet: Generation of ‘T v T’ fratricide resistant CAR T cells. A) Schema of base editing for T cells employing 3 rd generation codon optimised cytidine base deaminase (coBE3) fused to deactivated D10A Cas9 nickase and uracil glycosylase inhibitor (UGI) delivered as mRNA along with TRBC and CD7 single guide RNA (sgRNA). C- > U- > T conversion (G- > A antisense strand) resulting in STOP codon. B) Lentiviral transduction of edited cells from step 1 using 3 rd generation lentiviral vectors. Lentiviral plasmid configuration of CD3ε targeting 2 nd generation chimeric antigen receptor comprising OKT3 vL and vH scFv sequence fused to CD8 transmembrane domain (TM), 41BB co-stimulatory and CD3z activation domains under the control of a hPGK promoter. Lentiviral plasmid configuration of CD7 targeting 2 nd generation CAR comprising 3A1e vL and vH scFv sequence fused to CD8TM-41BB-CD3z under the control of a hPGK. C) coBE3 edited T cells devoid of shared antigens TCR/CD3 and CD7 surface receptors expressing either 3CAR or 7CAR evade fratricide and target T-ALL. BE: base editor; APOBEC: (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like); sgRNA: single guide RNA; PAM: protospacer adjacent motif; LTR: long terminal repeat; CMV: cytomegalovirus promoter; CAR: chimeric antigen receptor; cPPT: central polypurine track; U5: untranslated 5’ region; DU3: delta untranslated 3’ region; hPGK: human phosphoglycerate kinase promoter; vL: variable light chain; vH: variable heavy chain.

Techniques Used: Transduction, Plasmid Preparation, Sequencing, Activation Assay, Expressing

Cytidine deamination does not compromise integrity of antigen specificity of CAR sequences Serial examination of 3CAR or 7CAR scFv RNA sequences 48hrs and 96hrs after electroporation with SpCas9 (SP3/SP7) or coBE3 (BE3/BE7) mRNA and again at end of production on d14. A) Amplicons of 3CAR (left) and 7CAR (right) vH and vL sequences with antigen binding regions (ABR) displayed mapped as a Heatmap in R using the gplots library for C > N conversion rates at the marked sites. B) 3CAR (left) and 7CAR (right) scFv ABR mapped as a Heatmap for C > T conversion rates. C) Stacked histogram showing
Figure Legend Snippet: Cytidine deamination does not compromise integrity of antigen specificity of CAR sequences Serial examination of 3CAR or 7CAR scFv RNA sequences 48hrs and 96hrs after electroporation with SpCas9 (SP3/SP7) or coBE3 (BE3/BE7) mRNA and again at end of production on d14. A) Amplicons of 3CAR (left) and 7CAR (right) vH and vL sequences with antigen binding regions (ABR) displayed mapped as a Heatmap in R using the gplots library for C > N conversion rates at the marked sites. B) 3CAR (left) and 7CAR (right) scFv ABR mapped as a Heatmap for C > T conversion rates. C) Stacked histogram showing

Techniques Used: Electroporation, Binding Assay

7) Product Images from "Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions"

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

Journal: Oncotarget

doi: 10.18632/oncotarget.23453

Effects of estradiol on translocation of estrogen receptor alpha (ERα) to mitochondria Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye (middle panel). Merged signals indicated that the ERα protein had been translocated into mitochondria (bottom panels). These fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p
Figure Legend Snippet: Effects of estradiol on translocation of estrogen receptor alpha (ERα) to mitochondria Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye (middle panel). Merged signals indicated that the ERα protein had been translocated into mitochondria (bottom panels). These fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p

Techniques Used: Translocation Assay, Staining

Effects of estradiol on translocation of estrogen receptor alpha (ERα) to nuclei Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Cellular nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals indicated that the ERα protein had been translocated into nuclei (bottom panel). These merged fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p
Figure Legend Snippet: Effects of estradiol on translocation of estrogen receptor alpha (ERα) to nuclei Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Cellular nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals indicated that the ERα protein had been translocated into nuclei (bottom panel). These merged fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p

Techniques Used: Translocation Assay, Staining

8) Product Images from "Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis"

Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109352

Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
Figure Legend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.
Figure Legend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

Techniques Used: Immunofluorescence, Staining, Cell Culture

9) Product Images from "Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis"

Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109352

Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
Figure Legend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.
Figure Legend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

Techniques Used: Immunofluorescence, Staining, Cell Culture

10) Product Images from "DLBCL Cells with Acquired Resistance to Venetoclax Are Not Sensitized to BIRD-2 But Can Be Resensitized to Venetoclax through Bcl-XL Inhibition"

Article Title: DLBCL Cells with Acquired Resistance to Venetoclax Are Not Sensitized to BIRD-2 But Can Be Resensitized to Venetoclax through Bcl-XL Inhibition

Journal: Biomolecules

doi: 10.3390/biom10071081

Cytosolic IgG/IgM Ca 2+ responses and ER Ca 2+ content do not differ between Riva WT and Riva VR. ( a ) Typical cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT and VR. Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were stimulated with vehicle or 12 μg/mL IgG/IgM to provoke a cytosolic Ca 2+ signal. ( b ) Analysis of the area under the curve (AUC) of the IgG/IgM-triggered peak. Data are presented as average ± SEM (N = 5). ( c ) Cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT (black) and VR (red). Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were treated with thapsigargin (1 μM) to deplete ER Ca 2+ stores. Data are presented as average ± SEM (N = 6).
Figure Legend Snippet: Cytosolic IgG/IgM Ca 2+ responses and ER Ca 2+ content do not differ between Riva WT and Riva VR. ( a ) Typical cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT and VR. Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were stimulated with vehicle or 12 μg/mL IgG/IgM to provoke a cytosolic Ca 2+ signal. ( b ) Analysis of the area under the curve (AUC) of the IgG/IgM-triggered peak. Data are presented as average ± SEM (N = 5). ( c ) Cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT (black) and VR (red). Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were treated with thapsigargin (1 μM) to deplete ER Ca 2+ stores. Data are presented as average ± SEM (N = 6).

Techniques Used:

11) Product Images from "Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis"

Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109352

Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
Figure Legend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.
Figure Legend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

Techniques Used: Immunofluorescence, Staining, Cell Culture

12) Product Images from "Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia"

Article Title: Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20130110

IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.
Figure Legend Snippet: IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.

Techniques Used: Immunohistochemistry, Staining, In Vitro, Activity Assay, Construct, Sequencing, Clone Assay, Plasmid Preparation, Generated, Transduction, Flow Cytometry, Cytometry, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

13) Product Images from "Motherhood and infant contact regulate neuroplasticity in the serotonergic midbrain dorsal raphe"

Article Title: Motherhood and infant contact regulate neuroplasticity in the serotonergic midbrain dorsal raphe

Journal: Psychoneuroendocrinology

doi: 10.1016/j.psyneuen.2016.10.023

Newborn cell proliferation and survival in the DR of virgin and reproductive female rats A) Representative photomicrograph of BrdU-ir nuclei lining the lateral border of the cerebral aqueduct (aq). Scale bar = 50 μm. B) Photomicrograph showing BrdU-ir nuclei lining the lateral border of the aq. Note that no NeuN immunoreactivity is seen in this proliferative niche, where mature neurons do not reside. Scale bar = 50 μm. C) Representative photomicrograph of a pair of BrdU-ir nuclei in the DR. Scale bar = 20 μm. D) Representative photomicrograph of BrdU immunofluorescence in the DR, bordered by the medial longitudinal fasciculi (mlf). E) Representative photomicrograph of NeuN immunofluorescence in the DR. F) Overlay of cells containing BrdU immunofluorescence (green), NeuN immunofluorescence (red), and coliocalization (yellow) in the DR. Arrows indicate some colocalized nuclei. G) Effects of reproductive state on DR cell proliferation. H) Effects of reproductive state on newborn cells survival. Bars show means + SEMs. Unique letters above bars indicate statistically significant differences between groups, p
Figure Legend Snippet: Newborn cell proliferation and survival in the DR of virgin and reproductive female rats A) Representative photomicrograph of BrdU-ir nuclei lining the lateral border of the cerebral aqueduct (aq). Scale bar = 50 μm. B) Photomicrograph showing BrdU-ir nuclei lining the lateral border of the aq. Note that no NeuN immunoreactivity is seen in this proliferative niche, where mature neurons do not reside. Scale bar = 50 μm. C) Representative photomicrograph of a pair of BrdU-ir nuclei in the DR. Scale bar = 20 μm. D) Representative photomicrograph of BrdU immunofluorescence in the DR, bordered by the medial longitudinal fasciculi (mlf). E) Representative photomicrograph of NeuN immunofluorescence in the DR. F) Overlay of cells containing BrdU immunofluorescence (green), NeuN immunofluorescence (red), and coliocalization (yellow) in the DR. Arrows indicate some colocalized nuclei. G) Effects of reproductive state on DR cell proliferation. H) Effects of reproductive state on newborn cells survival. Bars show means + SEMs. Unique letters above bars indicate statistically significant differences between groups, p

Techniques Used: Immunofluorescence

14) Product Images from "Exercise‐induced α‐ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1‐dependent adrenal activation"

Article Title: Exercise‐induced α‐ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1‐dependent adrenal activation

Journal: The EMBO Journal

doi: 10.15252/embj.2019103304

AKG increases fat thermogenesis and lipolysis Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Body temperature of male mice after 11 weeks of AKG supplementation ( n = 9 per group). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6‐h cold exposure at 4°C in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of thermogenic genes (F) and immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation ( n = 3–6 per group). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks ( n = 9 per group). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1, and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks ( n = 8 per group). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Immunoblots (N) and quantification (O) of p‐HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation ( n = 3 per group). Representative images (P) and quantification (Q) of p‐HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation ( n = 9 per group). The mRNA expression of PPARγ, FASN, and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks ( n = 6 per group). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks ( n = 8–9 per group). Data information: Results are presented as mean ± SEM. In (B, C, E–G, I–K, M, O and Q–V), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by non‐paired Student's t ‐test.
Figure Legend Snippet: AKG increases fat thermogenesis and lipolysis Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Body temperature of male mice after 11 weeks of AKG supplementation ( n = 9 per group). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6‐h cold exposure at 4°C in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of thermogenic genes (F) and immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation ( n = 3–6 per group). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks ( n = 9 per group). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1, and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks ( n = 8 per group). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Immunoblots (N) and quantification (O) of p‐HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation ( n = 3 per group). Representative images (P) and quantification (Q) of p‐HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation ( n = 9 per group). The mRNA expression of PPARγ, FASN, and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks ( n = 6 per group). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks ( n = 8–9 per group). Data information: Results are presented as mean ± SEM. In (B, C, E–G, I–K, M, O and Q–V), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by non‐paired Student's t ‐test.

Techniques Used: Mouse Assay, Expressing, Western Blot, Staining

OXGR1 is required for metabolic beneficial effects of resistance exercise Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups receiving non‐exercise or resistance exercise for 14 days ( n = 8 per group). Exercise‐induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of the non‐exercise control group for each genotype ( n = 8 per group). Exercise‐induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice was subtracted by the average fat mass of the non‐exercise control group for each genotype ( n = 8 per group). Cumulative food intake of male WT littermates and OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Body composition of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks old) fed with normal chow were receiving resistance exercise for 40 min ( n = 8 per group). The serum AKG levels were tested before and immediately after exercise. Serum E level in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). The mRNA expression (J) and protein expression of UCP1 (K) in the BAT or the mRNA expression of HSL and ATGL (L) in the gWAT of male OXGR1KO mice after 14‐day resistance exercise ( n = 4 per group). Oxygen consumption (M, N) and RER (O, P) in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A–D) * P ≤ 0.05 and ** P ≤ 0.01 by two‐way ANOVA followed by post hoc Bonferroni tests. In (E–L, N and P), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests.
Figure Legend Snippet: OXGR1 is required for metabolic beneficial effects of resistance exercise Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups receiving non‐exercise or resistance exercise for 14 days ( n = 8 per group). Exercise‐induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of the non‐exercise control group for each genotype ( n = 8 per group). Exercise‐induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice was subtracted by the average fat mass of the non‐exercise control group for each genotype ( n = 8 per group). Cumulative food intake of male WT littermates and OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Body composition of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks old) fed with normal chow were receiving resistance exercise for 40 min ( n = 8 per group). The serum AKG levels were tested before and immediately after exercise. Serum E level in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). The mRNA expression (J) and protein expression of UCP1 (K) in the BAT or the mRNA expression of HSL and ATGL (L) in the gWAT of male OXGR1KO mice after 14‐day resistance exercise ( n = 4 per group). Oxygen consumption (M, N) and RER (O, P) in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A–D) * P ≤ 0.05 and ** P ≤ 0.01 by two‐way ANOVA followed by post hoc Bonferroni tests. In (E–L, N and P), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests.

Techniques Used: Mouse Assay, Expressing

Metabolic effects of AKG are mediated by adrenergic stimulation of thermogenesis and lipolysis Serum AKG concentration–time profile obtained from male C57BL/6 mice (10 weeks old) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level was tested at 0, 1, 2, 4, and 6 h after injection ( n = 8 per group). Representative images (B) and quantification (C) of BAT thermogenesis after 6‐h cold exposure at 4°C. Male C57BL/6 mice (10 weeks old) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4°C ( n = 8 per group). Immunoblots and quantification of p‐HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of 10 mg/kg AKG or saline ( n = 3 per group). Serum E level in AKG‐treated male mice 3 h after i.p. injection ( n = 8 per group). Oxygen consumption (F‐G) and RER (H‐I) in male C57BL/6 mice (10 weeks old) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor), or AKG + SR59230A ( n = 8 per group). All injections were performed at 7:00 am of the second day. Data are summarized in bar graph (G and I) by light or dark cycle of the second day. Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M), and serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks ( n = 8 per group). Representative images (O) and quantification (P) of BAT thermogenesis after 6‐h cold exposure at 4°C in sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 8 per group). The mRNA expression of thermogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 6 per group). Immunoblots (R) and quantification (S) of p‐HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Data information: Results are presented as mean ± SEM. In (A), * P ≤ 0.05 by non‐paired Student's t ‐test compared with before injection. In (C–E), * P ≤ 0.05 and ** P ≤ 0.01 by non‐paired Student's t ‐test. In (J, K), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests. In (G, I, L–N, P, Q, S and U), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests. Source data are available online for this figure.
Figure Legend Snippet: Metabolic effects of AKG are mediated by adrenergic stimulation of thermogenesis and lipolysis Serum AKG concentration–time profile obtained from male C57BL/6 mice (10 weeks old) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level was tested at 0, 1, 2, 4, and 6 h after injection ( n = 8 per group). Representative images (B) and quantification (C) of BAT thermogenesis after 6‐h cold exposure at 4°C. Male C57BL/6 mice (10 weeks old) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4°C ( n = 8 per group). Immunoblots and quantification of p‐HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of 10 mg/kg AKG or saline ( n = 3 per group). Serum E level in AKG‐treated male mice 3 h after i.p. injection ( n = 8 per group). Oxygen consumption (F‐G) and RER (H‐I) in male C57BL/6 mice (10 weeks old) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor), or AKG + SR59230A ( n = 8 per group). All injections were performed at 7:00 am of the second day. Data are summarized in bar graph (G and I) by light or dark cycle of the second day. Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M), and serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks ( n = 8 per group). Representative images (O) and quantification (P) of BAT thermogenesis after 6‐h cold exposure at 4°C in sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 8 per group). The mRNA expression of thermogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 6 per group). Immunoblots (R) and quantification (S) of p‐HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Data information: Results are presented as mean ± SEM. In (A), * P ≤ 0.05 by non‐paired Student's t ‐test compared with before injection. In (C–E), * P ≤ 0.05 and ** P ≤ 0.01 by non‐paired Student's t ‐test. In (J, K), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests. In (G, I, L–N, P, Q, S and U), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests. Source data are available online for this figure.

Techniques Used: Concentration Assay, Mouse Assay, Injection, Western Blot, Expressing

15) Product Images from "Exercise‐induced α‐ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1‐dependent adrenal activation"

Article Title: Exercise‐induced α‐ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1‐dependent adrenal activation

Journal: The EMBO Journal

doi: 10.15252/embj.2019103304

Acute in vivo effects of AKG Serum levels of NE (A) and NEFA (B) in male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 5–6 per group). The mRNA expression of thermogenic genes in male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 5–6 per group). Immunoblots and quantification of UCP1 in BAT of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 3 per group). Immunoblots and quantification of PLCβ and p‐Erk in the adrenal glands of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 3 per group). Physical activity (pedometer; F, G) and heart rate (H, I) of male mice i.p. injected with 10 mg/kg AKG or saline at 7:00 am ( n = 8 per group). Blood pressure of male mice i.p. injected with 10 mg/kg AKG or saline ( n = 8 per group). Serum levels of succinate (SUC) (M), fumaric acid (FUMA) (N), pyruvic acid (Pyr) (O), oxaloacetic acid (OAA) (P), α‐ketoleucine (α‐kehex) (Q), alpha‐ketoisovaleric acid (α‐keval) (R), and 2‐hydroxy‐3‐methylbutyric acid (2H3MA) (S) in male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A–E, G, I, L, N and Q), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.01 by non‐paired Student's t ‐test. In (F, H, J and K), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests.
Figure Legend Snippet: Acute in vivo effects of AKG Serum levels of NE (A) and NEFA (B) in male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 5–6 per group). The mRNA expression of thermogenic genes in male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 5–6 per group). Immunoblots and quantification of UCP1 in BAT of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 3 per group). Immunoblots and quantification of PLCβ and p‐Erk in the adrenal glands of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 3 per group). Physical activity (pedometer; F, G) and heart rate (H, I) of male mice i.p. injected with 10 mg/kg AKG or saline at 7:00 am ( n = 8 per group). Blood pressure of male mice i.p. injected with 10 mg/kg AKG or saline ( n = 8 per group). Serum levels of succinate (SUC) (M), fumaric acid (FUMA) (N), pyruvic acid (Pyr) (O), oxaloacetic acid (OAA) (P), α‐ketoleucine (α‐kehex) (Q), alpha‐ketoisovaleric acid (α‐keval) (R), and 2‐hydroxy‐3‐methylbutyric acid (2H3MA) (S) in male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of saline or AKG (10 mg/kg) ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A–E, G, I, L, N and Q), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.01 by non‐paired Student's t ‐test. In (F, H, J and K), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests.

Techniques Used: In Vivo, Mouse Assay, Injection, Expressing, Western Blot, Activity Assay

AKG increases fat thermogenesis and lipolysis Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Body temperature of male mice after 11 weeks of AKG supplementation ( n = 9 per group). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6‐h cold exposure at 4°C in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of thermogenic genes (F) and immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation ( n = 3–6 per group). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks ( n = 9 per group). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1, and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks ( n = 8 per group). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Immunoblots (N) and quantification (O) of p‐HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation ( n = 3 per group). Representative images (P) and quantification (Q) of p‐HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation ( n = 9 per group). The mRNA expression of PPARγ, FASN, and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks ( n = 6 per group). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks ( n = 8–9 per group). Data information: Results are presented as mean ± SEM. In (B, C, E–G, I–K, M, O and Q–V), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by non‐paired Student's t ‐test.
Figure Legend Snippet: AKG increases fat thermogenesis and lipolysis Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Body temperature of male mice after 11 weeks of AKG supplementation ( n = 9 per group). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6‐h cold exposure at 4°C in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of thermogenic genes (F) and immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation ( n = 3–6 per group). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks ( n = 9 per group). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks ( n = 9 per group). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1, and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks ( n = 8 per group). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation ( n = 8 per group). Immunoblots (N) and quantification (O) of p‐HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation ( n = 3 per group). Representative images (P) and quantification (Q) of p‐HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation ( n = 9 per group). The mRNA expression of PPARγ, FASN, and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks ( n = 6 per group). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks ( n = 8–9 per group). Data information: Results are presented as mean ± SEM. In (B, C, E–G, I–K, M, O and Q–V), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 by non‐paired Student's t ‐test.

Techniques Used: Mouse Assay, Expressing, Western Blot, Staining

OXGR1 is required for metabolic beneficial effects of resistance exercise Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups receiving non‐exercise or resistance exercise for 14 days ( n = 8 per group). Exercise‐induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of the non‐exercise control group for each genotype ( n = 8 per group). Exercise‐induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice was subtracted by the average fat mass of the non‐exercise control group for each genotype ( n = 8 per group). Cumulative food intake of male WT littermates and OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Body composition of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks old) fed with normal chow were receiving resistance exercise for 40 min ( n = 8 per group). The serum AKG levels were tested before and immediately after exercise. Serum E level in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). The mRNA expression (J) and protein expression of UCP1 (K) in the BAT or the mRNA expression of HSL and ATGL (L) in the gWAT of male OXGR1KO mice after 14‐day resistance exercise ( n = 4 per group). Oxygen consumption (M, N) and RER (O, P) in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A–D) * P ≤ 0.05 and ** P ≤ 0.01 by two‐way ANOVA followed by post hoc Bonferroni tests. In (E–L, N and P), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests.
Figure Legend Snippet: OXGR1 is required for metabolic beneficial effects of resistance exercise Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups receiving non‐exercise or resistance exercise for 14 days ( n = 8 per group). Exercise‐induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of the non‐exercise control group for each genotype ( n = 8 per group). Exercise‐induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice was subtracted by the average fat mass of the non‐exercise control group for each genotype ( n = 8 per group). Cumulative food intake of male WT littermates and OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Body composition of male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks old) fed with normal chow were receiving resistance exercise for 40 min ( n = 8 per group). The serum AKG levels were tested before and immediately after exercise. Serum E level in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). The mRNA expression (J) and protein expression of UCP1 (K) in the BAT or the mRNA expression of HSL and ATGL (L) in the gWAT of male OXGR1KO mice after 14‐day resistance exercise ( n = 4 per group). Oxygen consumption (M, N) and RER (O, P) in male OXGR1KO mice after 14‐day resistance exercise ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A–D) * P ≤ 0.05 and ** P ≤ 0.01 by two‐way ANOVA followed by post hoc Bonferroni tests. In (E–L, N and P), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests.

Techniques Used: Mouse Assay, Expressing

Metabolic effects of AKG are mediated by adrenergic stimulation of thermogenesis and lipolysis Serum AKG concentration–time profile obtained from male C57BL/6 mice (10 weeks old) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level was tested at 0, 1, 2, 4, and 6 h after injection ( n = 8 per group). Representative images (B) and quantification (C) of BAT thermogenesis after 6‐h cold exposure at 4°C. Male C57BL/6 mice (10 weeks old) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4°C ( n = 8 per group). Immunoblots and quantification of p‐HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of 10 mg/kg AKG or saline ( n = 3 per group). Serum E level in AKG‐treated male mice 3 h after i.p. injection ( n = 8 per group). Oxygen consumption (F‐G) and RER (H‐I) in male C57BL/6 mice (10 weeks old) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor), or AKG + SR59230A ( n = 8 per group). All injections were performed at 7:00 am of the second day. Data are summarized in bar graph (G and I) by light or dark cycle of the second day. Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M), and serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks ( n = 8 per group). Representative images (O) and quantification (P) of BAT thermogenesis after 6‐h cold exposure at 4°C in sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 8 per group). The mRNA expression of thermogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 6 per group). Immunoblots (R) and quantification (S) of p‐HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Data information: Results are presented as mean ± SEM. In (A), * P ≤ 0.05 by non‐paired Student's t ‐test compared with before injection. In (C–E), * P ≤ 0.05 and ** P ≤ 0.01 by non‐paired Student's t ‐test. In (J, K), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests. In (G, I, L–N, P, Q, S and U), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests. Source data are available online for this figure.
Figure Legend Snippet: Metabolic effects of AKG are mediated by adrenergic stimulation of thermogenesis and lipolysis Serum AKG concentration–time profile obtained from male C57BL/6 mice (10 weeks old) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level was tested at 0, 1, 2, 4, and 6 h after injection ( n = 8 per group). Representative images (B) and quantification (C) of BAT thermogenesis after 6‐h cold exposure at 4°C. Male C57BL/6 mice (10 weeks old) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4°C ( n = 8 per group). Immunoblots and quantification of p‐HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks old) 3 h after i.p. injection of 10 mg/kg AKG or saline ( n = 3 per group). Serum E level in AKG‐treated male mice 3 h after i.p. injection ( n = 8 per group). Oxygen consumption (F‐G) and RER (H‐I) in male C57BL/6 mice (10 weeks old) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor), or AKG + SR59230A ( n = 8 per group). All injections were performed at 7:00 am of the second day. Data are summarized in bar graph (G and I) by light or dark cycle of the second day. Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M), and serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks ( n = 8 per group). Representative images (O) and quantification (P) of BAT thermogenesis after 6‐h cold exposure at 4°C in sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 8 per group). The mRNA expression of thermogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 6 per group). Immunoblots (R) and quantification (S) of p‐HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group). Data information: Results are presented as mean ± SEM. In (A), * P ≤ 0.05 by non‐paired Student's t ‐test compared with before injection. In (C–E), * P ≤ 0.05 and ** P ≤ 0.01 by non‐paired Student's t ‐test. In (J, K), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests. In (G, I, L–N, P, Q, S and U), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests. Source data are available online for this figure.

Techniques Used: Concentration Assay, Mouse Assay, Injection, Western Blot, Expressing

Metabolic effects of AKG in mice fed on chow Body weight gain (A) and cumulative food intake (B) of male C57BL/6 mice. At 12 weeks of age, chow‐fed male mice were divided into two groups receiving tap water or water supplemented with 2% AKG for 6 weeks ( n = 8 per group). Body composition (C) and tissue weight (D) of male mice treated with AKG for 6 weeks ( n = 7–8 per group). Representative images (E) and quantification (F) of gWAT HE staining from male mice treated with AKG for 6 weeks ( n = 8 per group). Body weight gain (G) and cumulative food intake (H) of female C57BL/6 mice. At 12 weeks of age, chow‐fed female mice were divided into two groups receiving tap water or water supplemented with 2% AKG for 11 weeks ( n = 8 per group). Body composition (I) and tissue weight (J) of female mice treated with AKG for 11 weeks ( n = 8 per group). Representative images (K) and quantification (L) of gWAT HE staining from female mice treated with AKG for 11 weeks ( n = 8 per group). The mRNA expression of thermogenic genes in BAT of male C57BL/6 mice supplemented with AKG for 6 weeks ( n = 6 per group). Immunoblots and quantification of UCP1 (N) and representative images of DAB staining (O) and quantification (P) of UCP1 in BAT of male mice supplemented with AKG for 6 weeks ( n = 3–6 per group). Serum levels of NEFA (Q), E (R), NE (S), T3 (T), and T4 (U) in male mice supplemented with AKG for 6 weeks ( n = 6 per group). Data information: Results are presented as mean ± SEM. In (A, B, G and H), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests. In (C, D, F, I, J, L–N and P–U), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.01 by non‐paired Student's t ‐test. Source data are available online for this figure.
Figure Legend Snippet: Metabolic effects of AKG in mice fed on chow Body weight gain (A) and cumulative food intake (B) of male C57BL/6 mice. At 12 weeks of age, chow‐fed male mice were divided into two groups receiving tap water or water supplemented with 2% AKG for 6 weeks ( n = 8 per group). Body composition (C) and tissue weight (D) of male mice treated with AKG for 6 weeks ( n = 7–8 per group). Representative images (E) and quantification (F) of gWAT HE staining from male mice treated with AKG for 6 weeks ( n = 8 per group). Body weight gain (G) and cumulative food intake (H) of female C57BL/6 mice. At 12 weeks of age, chow‐fed female mice were divided into two groups receiving tap water or water supplemented with 2% AKG for 11 weeks ( n = 8 per group). Body composition (I) and tissue weight (J) of female mice treated with AKG for 11 weeks ( n = 8 per group). Representative images (K) and quantification (L) of gWAT HE staining from female mice treated with AKG for 11 weeks ( n = 8 per group). The mRNA expression of thermogenic genes in BAT of male C57BL/6 mice supplemented with AKG for 6 weeks ( n = 6 per group). Immunoblots and quantification of UCP1 (N) and representative images of DAB staining (O) and quantification (P) of UCP1 in BAT of male mice supplemented with AKG for 6 weeks ( n = 3–6 per group). Serum levels of NEFA (Q), E (R), NE (S), T3 (T), and T4 (U) in male mice supplemented with AKG for 6 weeks ( n = 6 per group). Data information: Results are presented as mean ± SEM. In (A, B, G and H), * P ≤ 0.05 by two‐way ANOVA followed by post hoc Bonferroni tests. In (C, D, F, I, J, L–N and P–U), * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.01 by non‐paired Student's t ‐test. Source data are available online for this figure.

Techniques Used: Mouse Assay, Staining, Expressing, Western Blot

Effects of AKG supplementation on fecal microbiota composition and iWAT browning in male mice Fecal energy of male C57BL/6 mice after 1, 4, and 11 weeks of AKG supplementation. At 12 weeks of age, male C57BL/6 mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks ( n = 9 per group). The fecal microbial composition in the phylum (B) and genus (C) in male C57BL/6 mice receiving 2% AKG water supplementation for 1 weeks (AKG1) or 4 weeks (AKG2) ( n = 5 per group). Community structure test by ANOSIM and ADONIS of beta diversity in the genus between groups. Immunoblots (E) and quantification (F) of UCP1 protein in the iWAT of male C57BL/6 mice. At 10 weeks of age, male C57BL/6 mice were switched from chow to HFD and divided into four groups receiving tap water + room temperature (RT, 23°C), tap water + cold exposure (6°C), 2% AKG supplementation + 23°C, and 2% AKG supplementation + 6°C for one week ( n = 8 per group). Representative images (G) and quantification (H, I) of HE staining or UCP1 staining in iWAT of male C57BL/6 mice ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A), data are analyzed by two‐way ANOVA followed by post hoc Bonferroni tests. In (F, H and I), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests.
Figure Legend Snippet: Effects of AKG supplementation on fecal microbiota composition and iWAT browning in male mice Fecal energy of male C57BL/6 mice after 1, 4, and 11 weeks of AKG supplementation. At 12 weeks of age, male C57BL/6 mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks ( n = 9 per group). The fecal microbial composition in the phylum (B) and genus (C) in male C57BL/6 mice receiving 2% AKG water supplementation for 1 weeks (AKG1) or 4 weeks (AKG2) ( n = 5 per group). Community structure test by ANOSIM and ADONIS of beta diversity in the genus between groups. Immunoblots (E) and quantification (F) of UCP1 protein in the iWAT of male C57BL/6 mice. At 10 weeks of age, male C57BL/6 mice were switched from chow to HFD and divided into four groups receiving tap water + room temperature (RT, 23°C), tap water + cold exposure (6°C), 2% AKG supplementation + 23°C, and 2% AKG supplementation + 6°C for one week ( n = 8 per group). Representative images (G) and quantification (H, I) of HE staining or UCP1 staining in iWAT of male C57BL/6 mice ( n = 8 per group). Data information: Results are presented as mean ± SEM. In (A), data are analyzed by two‐way ANOVA followed by post hoc Bonferroni tests. In (F, H and I), different letters between bars indicate P ≤ 0.05 by one‐way ANOVA followed by post hoc Tukey's tests.

Techniques Used: Mouse Assay, Western Blot, Staining

16) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

17) Product Images from "Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus"

Article Title: Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus

Journal: Scientific Reports

doi: 10.1038/srep27623

GMR biosensor autoantigen microarrays. ( a ) Optical images of a GMR biosensor chip and a cartridge with a reaction well (left). The sensor chip measures 10 × 12 mm and consists of an array of 8 × 10 sensors (total 80 sensors). Each sensor size is 100 × 100 μm (right). ( b ) A schematic of assaying antibody reactivity to autoantigens (not to scale). (1) Autoantigens were printed on the surface of the chip’s sensors. (2) The sample was added to the reaction well, allowing antibodies to bind to their corresponding antigens. (3) After washing, species-specific, biotinylated anti-IgG antibodies were used as a secondary reagent. (4) Streptavidin-coated MNPs bind to the biotinylated detection antibodies, and the respective sensor detects stray field from the bound MNPs.
Figure Legend Snippet: GMR biosensor autoantigen microarrays. ( a ) Optical images of a GMR biosensor chip and a cartridge with a reaction well (left). The sensor chip measures 10 × 12 mm and consists of an array of 8 × 10 sensors (total 80 sensors). Each sensor size is 100 × 100 μm (right). ( b ) A schematic of assaying antibody reactivity to autoantigens (not to scale). (1) Autoantigens were printed on the surface of the chip’s sensors. (2) The sample was added to the reaction well, allowing antibodies to bind to their corresponding antigens. (3) After washing, species-specific, biotinylated anti-IgG antibodies were used as a secondary reagent. (4) Streptavidin-coated MNPs bind to the biotinylated detection antibodies, and the respective sensor detects stray field from the bound MNPs.

Techniques Used: Chromatin Immunoprecipitation

18) Product Images from "Bystander suppression of collagen-induced arthritis in mice fed ovalbumin"

Article Title: Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

Journal: Arthritis Research & Therapy

doi: 10.1186/ar1150

Effects on anti-bovine collagen type II (BCII) antibody responses in mice fed ovalbumin (OVA). Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with BCII or BCII mixed with OVA, emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. Numbers of IgG anti-BCII antibody-forming spleen cells (AFCs) (a) , and serum IgG (b) , IgG 1 (c) , and IgG 2a (d) anti-BCII antibody activity, 1 week after booster immunization. Each symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. * P
Figure Legend Snippet: Effects on anti-bovine collagen type II (BCII) antibody responses in mice fed ovalbumin (OVA). Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with BCII or BCII mixed with OVA, emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. Numbers of IgG anti-BCII antibody-forming spleen cells (AFCs) (a) , and serum IgG (b) , IgG 1 (c) , and IgG 2a (d) anti-BCII antibody activity, 1 week after booster immunization. Each symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. * P

Techniques Used: Mouse Assay, Activity Assay, MANN-WHITNEY

Effects on ovalbumin (OVA)-specific immune responses in mice fed OVA. Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with OVA or OVA mixed with bovine collagen type II (BCII) emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. (a) IgG anti-OVA antibody activity in serum 1 week after the booster immunization. Each circular symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. In vitro proliferation (b) and secretion of IFN-γ (c) , IL-4 (d) , and IL-10 (e) by splenocytes after OVA restimulation 1 week after the booster immunization. The proliferation results are presented as proliferation indexes (mean counts per minute [cpm] in triplicate cultures stimulated with OVA/mean cpm in triplicate control cultures). Bars represent mean ± standard error of the mean ( n = 7–9). * P
Figure Legend Snippet: Effects on ovalbumin (OVA)-specific immune responses in mice fed OVA. Mice were fed OVA or a standard diet for 7 days. One week after the last day on the OVA diet, the mice were immunized with OVA or OVA mixed with bovine collagen type II (BCII) emulsified in Freund's complete adjuvant. Three weeks later the mice were immunized again, with the same antigen(s) emulsified in Freund's incomplete adjuvant. (a) IgG anti-OVA antibody activity in serum 1 week after the booster immunization. Each circular symbol represents one mouse and the bars represent the median values. Data were compared using the Mann–Whitney U test. In vitro proliferation (b) and secretion of IFN-γ (c) , IL-4 (d) , and IL-10 (e) by splenocytes after OVA restimulation 1 week after the booster immunization. The proliferation results are presented as proliferation indexes (mean counts per minute [cpm] in triplicate cultures stimulated with OVA/mean cpm in triplicate control cultures). Bars represent mean ± standard error of the mean ( n = 7–9). * P

Techniques Used: Mouse Assay, Activity Assay, MANN-WHITNEY, In Vitro

19) Product Images from "The E3 ligase VHL promotes follicular helper T cell differentiation via glycolytic-epigenetic control"

Article Title: The E3 ligase VHL promotes follicular helper T cell differentiation via glycolytic-epigenetic control

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20190337

HIF-1α mediates VHL regulation of Tfh cell development. (A) Representative flow-cytometric analysis of CD44 + CD4 + T cells from WT, VHL cKO, and Vhl fl/fl Hif1a fl/fl CD4 Cre (DKO) mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 3, DKO group). (C) Representative flow-cytometric plots of total B220 + B cells from WT, VHL cKO, and DKO mice as in A. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ) cells (bottom row) in the spleen. (D) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 4, DKO group). (E) ELISA of LCMV-specific IgG in the sera from infected mice as in A ( n = 4 per group), presented as absorbance at 490 nm (A490). (F–H) RT-PCR analysis of quantification of LCMV copies in serum (F), spleen (G), and kidney (H) from WT, VHL cKO, and DKO mice 8 d after LCMV infection ( n = 3 or 4 per group). (I) Representative flow-cytometric plots of donor CD45.2 + CD4 + T cells obtained from WT CD45.1 + host mice receiving naive CD4 + T cells from WT, VHL cKO, and DKO SMARTA mice, followed by infection with LCMV and analysis 8 d after infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (J) Quantification of frequency (among CD45.2 + CD4 + T cells) and number (CD45.2 + CD4 + ) of Tfh cells and GC-Tfh cells of WT CD45.1 host mice as in I ( n = 3–5 per group). (K and L) Quantification of Bcl-6 geometric mean fluorescence intensity (gMFI) in WT or VHL cKO Tfh-like cells cultured in the absence or presence of px478 (K) or CoCl 2 (L). Each symbol (B, D, F–H, and J) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P
Figure Legend Snippet: HIF-1α mediates VHL regulation of Tfh cell development. (A) Representative flow-cytometric analysis of CD44 + CD4 + T cells from WT, VHL cKO, and Vhl fl/fl Hif1a fl/fl CD4 Cre (DKO) mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 3, DKO group). (C) Representative flow-cytometric plots of total B220 + B cells from WT, VHL cKO, and DKO mice as in A. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ) cells (bottom row) in the spleen. (D) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 4, DKO group). (E) ELISA of LCMV-specific IgG in the sera from infected mice as in A ( n = 4 per group), presented as absorbance at 490 nm (A490). (F–H) RT-PCR analysis of quantification of LCMV copies in serum (F), spleen (G), and kidney (H) from WT, VHL cKO, and DKO mice 8 d after LCMV infection ( n = 3 or 4 per group). (I) Representative flow-cytometric plots of donor CD45.2 + CD4 + T cells obtained from WT CD45.1 + host mice receiving naive CD4 + T cells from WT, VHL cKO, and DKO SMARTA mice, followed by infection with LCMV and analysis 8 d after infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (J) Quantification of frequency (among CD45.2 + CD4 + T cells) and number (CD45.2 + CD4 + ) of Tfh cells and GC-Tfh cells of WT CD45.1 host mice as in I ( n = 3–5 per group). (K and L) Quantification of Bcl-6 geometric mean fluorescence intensity (gMFI) in WT or VHL cKO Tfh-like cells cultured in the absence or presence of px478 (K) or CoCl 2 (L). Each symbol (B, D, F–H, and J) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P

Techniques Used: Flow Cytometry, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Cell Culture

VHL deficiency results in defective Tfh cell development and function. (A) Representative flow-cytometric plots of activated CD44 + CD4 + T cells from WT and CD4 Cre Vhl fl/fl (VHL cKO) mice 8 d after infection with LCMV. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 6 per group). (C) RT-PCR analysis of mRNA of Tfh cell–related genes in CD44 + CD4 + and CD44 − CD4 + T cells from WT and VHL cKO mice 8 d after LCMV infection; results were normalized to those of Actb mRNA (encoding β-actin). (D) Representative flow-cytometric plots of total B220 + B cells from WT and VHL cKO mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ; bottom row) cells in the spleen. (E) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in D ( n = 6 per group). (F) ELISA of LCMV-specific IgG in the sera from infected mice as in D ( n = 6 per group), presented as absorbance at 490 nm (A490). (G) Representative flow-cytometric plots of CD44 + CD4 + T cells from WT and VHL cKO mice 7 d after immunization with SRBC. Numbers adjacent to outlined areas indicate frequency of GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (H) Quantification of frequency (among CD44 + CD4 + T cells) and number of GC-Tfh cells of mice as in G ( n = 3–4 per group). Each symbol (B, E, and H) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P
Figure Legend Snippet: VHL deficiency results in defective Tfh cell development and function. (A) Representative flow-cytometric plots of activated CD44 + CD4 + T cells from WT and CD4 Cre Vhl fl/fl (VHL cKO) mice 8 d after infection with LCMV. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 6 per group). (C) RT-PCR analysis of mRNA of Tfh cell–related genes in CD44 + CD4 + and CD44 − CD4 + T cells from WT and VHL cKO mice 8 d after LCMV infection; results were normalized to those of Actb mRNA (encoding β-actin). (D) Representative flow-cytometric plots of total B220 + B cells from WT and VHL cKO mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ; bottom row) cells in the spleen. (E) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in D ( n = 6 per group). (F) ELISA of LCMV-specific IgG in the sera from infected mice as in D ( n = 6 per group), presented as absorbance at 490 nm (A490). (G) Representative flow-cytometric plots of CD44 + CD4 + T cells from WT and VHL cKO mice 7 d after immunization with SRBC. Numbers adjacent to outlined areas indicate frequency of GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (H) Quantification of frequency (among CD44 + CD4 + T cells) and number of GC-Tfh cells of mice as in G ( n = 3–4 per group). Each symbol (B, E, and H) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P

Techniques Used: Flow Cytometry, Mouse Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

20) Product Images from "UDP-Glucuronosyltransferase 1a Enzymes Are Present and Active in the Mouse Blastocyst"

Article Title: UDP-Glucuronosyltransferase 1a Enzymes Are Present and Active in the Mouse Blastocyst

Journal: Drug Metabolism and Disposition

doi: 10.1124/dmd.114.059766

Confocal immunofluorescence analysis of Ugt expression and localization in blastocysts. (A) Exemplary images of blastocysts stained with pan-specific antibodies against Ugt1a and Ugt2b, with DAPI staining to show the cell nuclei. Strong Ugt1a signal is
Figure Legend Snippet: Confocal immunofluorescence analysis of Ugt expression and localization in blastocysts. (A) Exemplary images of blastocysts stained with pan-specific antibodies against Ugt1a and Ugt2b, with DAPI staining to show the cell nuclei. Strong Ugt1a signal is

Techniques Used: Immunofluorescence, Expressing, Staining

21) Product Images from "Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer"

Article Title: Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0000000000000203

IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P
Figure Legend Snippet: IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P

Techniques Used: Expressing, Transduction, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P
Figure Legend Snippet: Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P

Techniques Used: Construct, Mouse Assay

Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.
Figure Legend Snippet: Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.

Techniques Used: Mouse Assay

22) Product Images from "Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells"

Article Title: Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells

Journal: Cancer Immunology, Immunotherapy

doi: 10.1007/s00262-016-1901-y

Proliferation of human iNKT cells with PDL1 blockade. PBMCs were obtained from six healthy donors and eight non-small cell lung cancer patients. On day 0, PBMCs were stimulated with αGalCer-pulsed IL-2/GM-CSF cultured APCs with anti-PDL1 antibody or isotype control. On day 7, cells were collected and restimulated with PDL1-blocked or isotype control-treated APCs at a ratio of 1:2.5. The cells were collected and counted on day 14, and the proportion of Vα24 + Vβ11 + iNKT cells was analyzed using flow cytometry. a Anti-PDL1 antibody binding and PDL1 positivity on APCs were assessed using anti-mouse biotin plus streptavidin staining. b The percentage of PDL1-positive iNKT cells on days 0 and 7 were analyzed with APC-conjugated anti-human PDL1. The gray-shaded histogram represents the isotype control; the unshaded histogram represents PDL1. c The number of Vα24 + Vβ11 + iNKT cells on day 7 is shown. PDL1 positivity on APCs was analyzed according to the population comparison method using the FlowJo software program. P values were calculated using the unpaired t test. isotype, isotype control; aPDL1 ab, anti-PDL1 antibody
Figure Legend Snippet: Proliferation of human iNKT cells with PDL1 blockade. PBMCs were obtained from six healthy donors and eight non-small cell lung cancer patients. On day 0, PBMCs were stimulated with αGalCer-pulsed IL-2/GM-CSF cultured APCs with anti-PDL1 antibody or isotype control. On day 7, cells were collected and restimulated with PDL1-blocked or isotype control-treated APCs at a ratio of 1:2.5. The cells were collected and counted on day 14, and the proportion of Vα24 + Vβ11 + iNKT cells was analyzed using flow cytometry. a Anti-PDL1 antibody binding and PDL1 positivity on APCs were assessed using anti-mouse biotin plus streptavidin staining. b The percentage of PDL1-positive iNKT cells on days 0 and 7 were analyzed with APC-conjugated anti-human PDL1. The gray-shaded histogram represents the isotype control; the unshaded histogram represents PDL1. c The number of Vα24 + Vβ11 + iNKT cells on day 7 is shown. PDL1 positivity on APCs was analyzed according to the population comparison method using the FlowJo software program. P values were calculated using the unpaired t test. isotype, isotype control; aPDL1 ab, anti-PDL1 antibody

Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Binding Assay, Staining, Software

23) Product Images from "DLBCL Cells with Acquired Resistance to Venetoclax Are Not Sensitized to BIRD-2 But Can Be Resensitized to Venetoclax through Bcl-XL Inhibition"

Article Title: DLBCL Cells with Acquired Resistance to Venetoclax Are Not Sensitized to BIRD-2 But Can Be Resensitized to Venetoclax through Bcl-XL Inhibition

Journal: Biomolecules

doi: 10.3390/biom10071081

Cytosolic IgG/IgM Ca 2+ responses and ER Ca 2+ content do not differ between Riva WT and Riva VR. ( a ) Typical cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT and VR. Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were stimulated with vehicle or 12 μg/mL IgG/IgM to provoke a cytosolic Ca 2+ signal. ( b ) Analysis of the area under the curve (AUC) of the IgG/IgM-triggered peak. Data are presented as average ± SEM (N = 5). ( c ) Cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT (black) and VR (red). Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were treated with thapsigargin (1 μM) to deplete ER Ca 2+ stores. Data are presented as average ± SEM (N = 6).
Figure Legend Snippet: Cytosolic IgG/IgM Ca 2+ responses and ER Ca 2+ content do not differ between Riva WT and Riva VR. ( a ) Typical cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT and VR. Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were stimulated with vehicle or 12 μg/mL IgG/IgM to provoke a cytosolic Ca 2+ signal. ( b ) Analysis of the area under the curve (AUC) of the IgG/IgM-triggered peak. Data are presented as average ± SEM (N = 5). ( c ) Cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT (black) and VR (red). Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were treated with thapsigargin (1 μM) to deplete ER Ca 2+ stores. Data are presented as average ± SEM (N = 6).

Techniques Used:

24) Product Images from "Base-edited CAR T Cells for combinational therapy against T cell malignancies"

Article Title: Base-edited CAR T Cells for combinational therapy against T cell malignancies

Journal: bioRxiv

doi: 10.1101/2020.07.30.228429

3CAR and 7CAR primary T cells evade fratricide during production. A) Phenotypic analysis of surface antigen CD3 and CD7 expression (top panel) and CAR expression (bottom panel) of CD3/CD28 activated peripheral blood mononuclear cells elcetroporated with sgRNA targeting TRBC and CD7 alongside coBE3 mRNA and subsequently transduced with 3CAR or 7CAR lentiviral vectors at MOI 5. Reduced TCR/CD3 and CD7 expression in edited groups and high level CAR expression with fratricide evasion (dotted red outline) was exhibited (n=4). Self-enrichment effects followed co-culture of 3CAR and 7CAR products (n=2) resulted in enriched TCR - CD7 - 3CAR/7CAR cells (red box). B) Proportion of CD3 or CD7 surface antigen and CAR expression at end of 3CAR (n=4), 7CAR (n=4) production or untransduced (UTD) (n=4) cells. Error bars represent SEM across (n=4) donors. C) Schematic of exonic regions within TRBC and CD7 genes. Red marking in exons 4 of TRBC and CD7 represent genomic translation stop sites followed by 5’ untranslated regions (white boxes). Red triangles with asterisk indicate position of base conversion resulting in premature stop codon formation. D) Representative base EDITR output of Sanger sequencing results from mixed 3CAR/7CAR co-culture DNA PCR amplicons of TRBC and CD7 genomic loci. Sites of intended base conversion highlighted in red boxes. High frequency G- > A (antisense) and C- > T changes within editing window highlighted by red vs blue colour. E) Percentage of G- > A conversions throughout TRBC-targeting protospacer sequence (left), and C- > T conversions throughout CD7-targeting protospacer sequence (right).
Figure Legend Snippet: 3CAR and 7CAR primary T cells evade fratricide during production. A) Phenotypic analysis of surface antigen CD3 and CD7 expression (top panel) and CAR expression (bottom panel) of CD3/CD28 activated peripheral blood mononuclear cells elcetroporated with sgRNA targeting TRBC and CD7 alongside coBE3 mRNA and subsequently transduced with 3CAR or 7CAR lentiviral vectors at MOI 5. Reduced TCR/CD3 and CD7 expression in edited groups and high level CAR expression with fratricide evasion (dotted red outline) was exhibited (n=4). Self-enrichment effects followed co-culture of 3CAR and 7CAR products (n=2) resulted in enriched TCR - CD7 - 3CAR/7CAR cells (red box). B) Proportion of CD3 or CD7 surface antigen and CAR expression at end of 3CAR (n=4), 7CAR (n=4) production or untransduced (UTD) (n=4) cells. Error bars represent SEM across (n=4) donors. C) Schematic of exonic regions within TRBC and CD7 genes. Red marking in exons 4 of TRBC and CD7 represent genomic translation stop sites followed by 5’ untranslated regions (white boxes). Red triangles with asterisk indicate position of base conversion resulting in premature stop codon formation. D) Representative base EDITR output of Sanger sequencing results from mixed 3CAR/7CAR co-culture DNA PCR amplicons of TRBC and CD7 genomic loci. Sites of intended base conversion highlighted in red boxes. High frequency G- > A (antisense) and C- > T changes within editing window highlighted by red vs blue colour. E) Percentage of G- > A conversions throughout TRBC-targeting protospacer sequence (left), and C- > T conversions throughout CD7-targeting protospacer sequence (right).

Techniques Used: Expressing, Transduction, Co-Culture Assay, Sequencing, Polymerase Chain Reaction

3CAR and 7CAR cells effectively clear T cell malignancy in vivo. A) Experimental timeline of GFP + LUC + Jurkat tumour injection (Day 0) and effector T cell injection (Day 4) in n=27 NOD/SCID/γc −/− (NSG) mice. Bioluminescent imaging (BLI) performed biweekly (Days 3-24). Organ harvest post mortem for flow-based characterisation (Day 24). B) NSG mice were infused with 1 x 10 7 GFP+LUC+ Jurkat T cells modified to express mixed CD3 and/or CD7 surface antigens in groups of (n=5) CD3 - CD7 - , (n=4) CD3 + CD7 - , (n=5) CD3 - CD7 + or (n=5) CD3 - CD7 - and imaged on day 3 prior to infusion of 1 x 10 7 TCR - CD7 - 3CAR/7CAR mixed effectors or untransduced (UTD) cells. Leukaemic progression monitored by serial BLI for 24 days and revealed disease progression in animals receiving untransduced T cells (3CAR - 7CAR - ) and in animals engrafted with antigen negative (CD3 - CD7 - ) leukemia. C) Bioluminescence signal of each animal plotted as Average radiance [photons/sec/cm 2 /sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent SEM. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc ****P
Figure Legend Snippet: 3CAR and 7CAR cells effectively clear T cell malignancy in vivo. A) Experimental timeline of GFP + LUC + Jurkat tumour injection (Day 0) and effector T cell injection (Day 4) in n=27 NOD/SCID/γc −/− (NSG) mice. Bioluminescent imaging (BLI) performed biweekly (Days 3-24). Organ harvest post mortem for flow-based characterisation (Day 24). B) NSG mice were infused with 1 x 10 7 GFP+LUC+ Jurkat T cells modified to express mixed CD3 and/or CD7 surface antigens in groups of (n=5) CD3 - CD7 - , (n=4) CD3 + CD7 - , (n=5) CD3 - CD7 + or (n=5) CD3 - CD7 - and imaged on day 3 prior to infusion of 1 x 10 7 TCR - CD7 - 3CAR/7CAR mixed effectors or untransduced (UTD) cells. Leukaemic progression monitored by serial BLI for 24 days and revealed disease progression in animals receiving untransduced T cells (3CAR - 7CAR - ) and in animals engrafted with antigen negative (CD3 - CD7 - ) leukemia. C) Bioluminescence signal of each animal plotted as Average radiance [photons/sec/cm 2 /sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent SEM. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc ****P

Techniques Used: In Vivo, Injection, Mouse Assay, Imaging, Modification

Multiplexed cytidine deamination reduces frequency of dsDNA break-mediated chromosomal translocations. A) Schematic of TRBC locus within chromosome 7 q-arm and CD7 locus within chromosome 17 q-arm highlighted by red line. Four predicted chromosomal translocations generated following simultaneous dsDNA-mediated cleavage at TRBC and CD7 loci. B) Gel electrophoresis of DNA products from either untransduced (UTD) or mixed 3CAR/7CAR co-cultures edited with spCas9 or coBE3 mRNA following PCR amplification with TRBC Fwd - CD7 Fwd, TRBC Rev – CD7 Fwd, TRBC Rev – CD7 Rev and TRBC Fwd- CD7 Rev primer combinations. Positive bands detected at ∼250bp. Control bands are PCR amplicons from of synthesised fusions. C) Histogram showing percentage of digital droplet PCR (ddPCR)-based quantification of four possible predicted translocations (T1-T4) in DNA from mixed 3CAR/7CAR co- cultures edited with spCas9 or coBE3 mRNA.
Figure Legend Snippet: Multiplexed cytidine deamination reduces frequency of dsDNA break-mediated chromosomal translocations. A) Schematic of TRBC locus within chromosome 7 q-arm and CD7 locus within chromosome 17 q-arm highlighted by red line. Four predicted chromosomal translocations generated following simultaneous dsDNA-mediated cleavage at TRBC and CD7 loci. B) Gel electrophoresis of DNA products from either untransduced (UTD) or mixed 3CAR/7CAR co-cultures edited with spCas9 or coBE3 mRNA following PCR amplification with TRBC Fwd - CD7 Fwd, TRBC Rev – CD7 Fwd, TRBC Rev – CD7 Rev and TRBC Fwd- CD7 Rev primer combinations. Positive bands detected at ∼250bp. Control bands are PCR amplicons from of synthesised fusions. C) Histogram showing percentage of digital droplet PCR (ddPCR)-based quantification of four possible predicted translocations (T1-T4) in DNA from mixed 3CAR/7CAR co- cultures edited with spCas9 or coBE3 mRNA.

Techniques Used: Generated, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification

3CAR/7CAR T cells mediate potent killing of T-ALL cells in vitro . In vitro cytotoxicity of 3CAR and 7CAR cells against T-ALL cell lines and primary T-ALL targets. A) 51 Cr labelled Jurkat T cells modified to express CD3 + CD7 + , CD3 + CD7 - , CD3 - CD7 + or CD3 - CD7 - were co-cultured with either 3CAR (white squares), 7CAR (grey squares), mixed 3CAR/7CAR (black squares) or untransduced (white circles) cells at an increasing ratio of effectors:targets (E:T). Error bars represent SEM of (n=3) technical replicates. B) Cytotoxic activity of 3CAR, 7CAR, mixed 3CAR/7CAR or untransduced primary T cell controls against primary patient T-ALL cells. Representative flow cytometry plots gated on CFSE + live T-ALL tumour cells (top panel). Frequency of surface antigens CD3, CD7 (middle panel) and CD19, CD56, (lower panel) gated on CFSE+ live tumour cells.
Figure Legend Snippet: 3CAR/7CAR T cells mediate potent killing of T-ALL cells in vitro . In vitro cytotoxicity of 3CAR and 7CAR cells against T-ALL cell lines and primary T-ALL targets. A) 51 Cr labelled Jurkat T cells modified to express CD3 + CD7 + , CD3 + CD7 - , CD3 - CD7 + or CD3 - CD7 - were co-cultured with either 3CAR (white squares), 7CAR (grey squares), mixed 3CAR/7CAR (black squares) or untransduced (white circles) cells at an increasing ratio of effectors:targets (E:T). Error bars represent SEM of (n=3) technical replicates. B) Cytotoxic activity of 3CAR, 7CAR, mixed 3CAR/7CAR or untransduced primary T cell controls against primary patient T-ALL cells. Representative flow cytometry plots gated on CFSE + live T-ALL tumour cells (top panel). Frequency of surface antigens CD3, CD7 (middle panel) and CD19, CD56, (lower panel) gated on CFSE+ live tumour cells.

Techniques Used: In Vitro, Modification, Cell Culture, Activity Assay, Flow Cytometry

Generation of ‘T v T’ fratricide resistant CAR T cells. A) Schema of base editing for T cells employing 3 rd generation codon optimised cytidine base deaminase (coBE3) fused to deactivated D10A Cas9 nickase and uracil glycosylase inhibitor (UGI) delivered as mRNA along with TRBC and CD7 single guide RNA (sgRNA). C- > U- > T conversion (G- > A antisense strand) resulting in STOP codon. B) Lentiviral transduction of edited cells from step 1 using 3 rd generation lentiviral vectors. Lentiviral plasmid configuration of CD3ε targeting 2 nd generation chimeric antigen receptor comprising OKT3 vL and vH scFv sequence fused to CD8 transmembrane domain (TM), 41BB co-stimulatory and CD3z activation domains under the control of a hPGK promoter. Lentiviral plasmid configuration of CD7 targeting 2 nd generation CAR comprising 3A1e vL and vH scFv sequence fused to CD8TM-41BB-CD3z under the control of a hPGK. C) coBE3 edited T cells devoid of shared antigens TCR/CD3 and CD7 surface receptors expressing either 3CAR or 7CAR evade fratricide and target T-ALL. BE: base editor; APOBEC: (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like); sgRNA: single guide RNA; PAM: protospacer adjacent motif; LTR: long terminal repeat; CMV: cytomegalovirus promoter; CAR: chimeric antigen receptor; cPPT: central polypurine track; U5: untranslated 5’ region; DU3: delta untranslated 3’ region; hPGK: human phosphoglycerate kinase promoter; vL: variable light chain; vH: variable heavy chain.
Figure Legend Snippet: Generation of ‘T v T’ fratricide resistant CAR T cells. A) Schema of base editing for T cells employing 3 rd generation codon optimised cytidine base deaminase (coBE3) fused to deactivated D10A Cas9 nickase and uracil glycosylase inhibitor (UGI) delivered as mRNA along with TRBC and CD7 single guide RNA (sgRNA). C- > U- > T conversion (G- > A antisense strand) resulting in STOP codon. B) Lentiviral transduction of edited cells from step 1 using 3 rd generation lentiviral vectors. Lentiviral plasmid configuration of CD3ε targeting 2 nd generation chimeric antigen receptor comprising OKT3 vL and vH scFv sequence fused to CD8 transmembrane domain (TM), 41BB co-stimulatory and CD3z activation domains under the control of a hPGK promoter. Lentiviral plasmid configuration of CD7 targeting 2 nd generation CAR comprising 3A1e vL and vH scFv sequence fused to CD8TM-41BB-CD3z under the control of a hPGK. C) coBE3 edited T cells devoid of shared antigens TCR/CD3 and CD7 surface receptors expressing either 3CAR or 7CAR evade fratricide and target T-ALL. BE: base editor; APOBEC: (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like); sgRNA: single guide RNA; PAM: protospacer adjacent motif; LTR: long terminal repeat; CMV: cytomegalovirus promoter; CAR: chimeric antigen receptor; cPPT: central polypurine track; U5: untranslated 5’ region; DU3: delta untranslated 3’ region; hPGK: human phosphoglycerate kinase promoter; vL: variable light chain; vH: variable heavy chain.

Techniques Used: Transduction, Plasmid Preparation, Sequencing, Activation Assay, Expressing

Cytidine deamination does not compromise integrity of antigen specificity of CAR sequences Serial examination of 3CAR or 7CAR scFv RNA sequences 48hrs and 96hrs after electroporation with SpCas9 (SP3/SP7) or coBE3 (BE3/BE7) mRNA and again at end of production on d14. A) Amplicons of 3CAR (left) and 7CAR (right) vH and vL sequences with antigen binding regions (ABR) displayed mapped as a Heatmap in R using the gplots library for C > N conversion rates at the marked sites. B) 3CAR (left) and 7CAR (right) scFv ABR mapped as a Heatmap for C > T conversion rates. C) Stacked histogram showing
Figure Legend Snippet: Cytidine deamination does not compromise integrity of antigen specificity of CAR sequences Serial examination of 3CAR or 7CAR scFv RNA sequences 48hrs and 96hrs after electroporation with SpCas9 (SP3/SP7) or coBE3 (BE3/BE7) mRNA and again at end of production on d14. A) Amplicons of 3CAR (left) and 7CAR (right) vH and vL sequences with antigen binding regions (ABR) displayed mapped as a Heatmap in R using the gplots library for C > N conversion rates at the marked sites. B) 3CAR (left) and 7CAR (right) scFv ABR mapped as a Heatmap for C > T conversion rates. C) Stacked histogram showing

Techniques Used: Electroporation, Binding Assay

25) Product Images from "Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts"

Article Title: Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts

Journal: Journal for Immunotherapy of Cancer

doi: 10.1186/2051-1426-1-21

Ectopic expression miR-17-92 in CAR-T cells confers improved protection following tumor re-challenge. (A) Schematic of the experimental protocol. CAR-T-cell-treated mice that survived at least for 35 days in the experiment shown in Figure 4 [4 mice receiving 3C10-T-cells without miR and 3 mice treated with T-cells co-transduced with 3C10 plus miR-17-92] received i.c. re-challenge with U87-EGFRvIII-Luc cells (5×10 5 /mice) on day 49. No additional CAR T-cells were injected. (B) Longitudinal measurements of tumor-derived mean photon flux ± SD from the 2 groups of mice. The background luminescence level (up to 10 3 p/s) was defined based on the levels observed in non-tumor-bearing mice imaged in parallel with tumor-bearing mice in treatment groups. *p
Figure Legend Snippet: Ectopic expression miR-17-92 in CAR-T cells confers improved protection following tumor re-challenge. (A) Schematic of the experimental protocol. CAR-T-cell-treated mice that survived at least for 35 days in the experiment shown in Figure 4 [4 mice receiving 3C10-T-cells without miR and 3 mice treated with T-cells co-transduced with 3C10 plus miR-17-92] received i.c. re-challenge with U87-EGFRvIII-Luc cells (5×10 5 /mice) on day 49. No additional CAR T-cells were injected. (B) Longitudinal measurements of tumor-derived mean photon flux ± SD from the 2 groups of mice. The background luminescence level (up to 10 3 p/s) was defined based on the levels observed in non-tumor-bearing mice imaged in parallel with tumor-bearing mice in treatment groups. *p

Techniques Used: Expressing, Mouse Assay, Transduction, Injection, Derivative Assay

Robust therapeutic effects of CAR-T-cells in mice bearing U87-EGFRvIII tumors. (A) Schematic of the experimental protocol. NSG mice received i.c. inoculation of 5×10 4 U87-EGFRvIII-Luc cells on day -7 and subsequently received a single i.v. infusion of 2×10 6 T cells transduced with pELNS-3C10-CAR alone or with both pELNS-3C10-CAR and FG12-EF1a-miR-17/92 or mock vector on day 0. All mice received i.p. administration of TMZ (0.33 mg/mouse/day) on days 0–4. (B) Kaplan-Meier analysis. Median survival of the mice treated with CAR-T cells (with or without co-transduction of miR-17-92; n = 10/group) was significantly greater compared with the mice treated with mock-transduced T cells (p
Figure Legend Snippet: Robust therapeutic effects of CAR-T-cells in mice bearing U87-EGFRvIII tumors. (A) Schematic of the experimental protocol. NSG mice received i.c. inoculation of 5×10 4 U87-EGFRvIII-Luc cells on day -7 and subsequently received a single i.v. infusion of 2×10 6 T cells transduced with pELNS-3C10-CAR alone or with both pELNS-3C10-CAR and FG12-EF1a-miR-17/92 or mock vector on day 0. All mice received i.p. administration of TMZ (0.33 mg/mouse/day) on days 0–4. (B) Kaplan-Meier analysis. Median survival of the mice treated with CAR-T cells (with or without co-transduction of miR-17-92; n = 10/group) was significantly greater compared with the mice treated with mock-transduced T cells (p

Techniques Used: Mouse Assay, Transduction, Plasmid Preparation

26) Product Images from "Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration"

Article Title: Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration

Journal: World Journal of Experimental Medicine

doi: 10.5493/wjem.v5.i2.140

Pax7 + cells in regenerating skeletal muscle implants. A, B: Examples of single (A and B) and pairs of (B) Pax7 + cells (arrows) attached or positioned in close proximity to myofibers in a fresh mouse implant at 14 d after implantation. Scale bar is 50
Figure Legend Snippet: Pax7 + cells in regenerating skeletal muscle implants. A, B: Examples of single (A and B) and pairs of (B) Pax7 + cells (arrows) attached or positioned in close proximity to myofibers in a fresh mouse implant at 14 d after implantation. Scale bar is 50

Techniques Used:

27) Product Images from "Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils"

Article Title: Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00973

Increased cytotoxicity of ferrous ion-exposed IVIG involves FAS signaling in neutrophils. (A) Concentration-dependent death response of autologous neutrophils to IVIG (native or ferrous ion-exposed) following preincubation with polymorphonuclear neutrophils (PMNs) (IVIG PMN ) or medium (IVIG medium ). (B) Neutrophil death upon preincubation of native or Fe(II)-IVIG with recombinant TNF-R1-, Siglec-9- or FAS-Fc proteins in presence of absence of GM-CSF or LPS. (C) Surface expression of FAS ligand (FASL) on unprimed or primed (GM-CSF or LPS) neutrophils upon culture for 15 h in medium, native or Fe(II)-IVIG. (D) Immunoblotting indicating native and Fe(II)-IVIG reactivity to immobilized recombinant FAS-Fc protein. (E) Blocking effect of IVIG preincubation at two different concentrations on neutrophil death induced by an α-FAS monoclonal antibody (mAb, clone CH11). The dashed line represents the mean level of neutrophil death specifically induced by α-FAS mAb treatment. Students t test (B) , paired t test (D) , one-way ANOVA, followed by Tukey's posttest for comparisons among groups (C) , or Dunnett's posttest with anti-FAS as control (E) . Data are representative of three (A) , four (D) five (C) , or six (B,E) independent experiments (mean ± SEM). * P
Figure Legend Snippet: Increased cytotoxicity of ferrous ion-exposed IVIG involves FAS signaling in neutrophils. (A) Concentration-dependent death response of autologous neutrophils to IVIG (native or ferrous ion-exposed) following preincubation with polymorphonuclear neutrophils (PMNs) (IVIG PMN ) or medium (IVIG medium ). (B) Neutrophil death upon preincubation of native or Fe(II)-IVIG with recombinant TNF-R1-, Siglec-9- or FAS-Fc proteins in presence of absence of GM-CSF or LPS. (C) Surface expression of FAS ligand (FASL) on unprimed or primed (GM-CSF or LPS) neutrophils upon culture for 15 h in medium, native or Fe(II)-IVIG. (D) Immunoblotting indicating native and Fe(II)-IVIG reactivity to immobilized recombinant FAS-Fc protein. (E) Blocking effect of IVIG preincubation at two different concentrations on neutrophil death induced by an α-FAS monoclonal antibody (mAb, clone CH11). The dashed line represents the mean level of neutrophil death specifically induced by α-FAS mAb treatment. Students t test (B) , paired t test (D) , one-way ANOVA, followed by Tukey's posttest for comparisons among groups (C) , or Dunnett's posttest with anti-FAS as control (E) . Data are representative of three (A) , four (D) five (C) , or six (B,E) independent experiments (mean ± SEM). * P

Techniques Used: Concentration Assay, Recombinant, Expressing, Blocking Assay

28) Product Images from "Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies"

Article Title: Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies

Journal: bioRxiv

doi: 10.1101/2020.06.02.131102

Proof-of-concept MInD-based SARS-CoV-2 specific antibody detection. IFCs were coated with commercial 2 μg·ml −1 SARS-CoV-2 spike protein peptide and blocked with BSA. A corresponding antibody was diluted either in PBS (black curves) or spiked in human serum (red curves) and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized magnetic particles. Assay time of this preliminary MInD setup was 42 minutes (without column preparation). Limit of detection (LOD) was determined by help of non-linear hill fit. Each data point represents mean ± SD (n = 2).
Figure Legend Snippet: Proof-of-concept MInD-based SARS-CoV-2 specific antibody detection. IFCs were coated with commercial 2 μg·ml −1 SARS-CoV-2 spike protein peptide and blocked with BSA. A corresponding antibody was diluted either in PBS (black curves) or spiked in human serum (red curves) and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized magnetic particles. Assay time of this preliminary MInD setup was 42 minutes (without column preparation). Limit of detection (LOD) was determined by help of non-linear hill fit. Each data point represents mean ± SD (n = 2).

Techniques Used:

Proof-of-concept MInD assay setup using IFC coated with SARS-CoV-2 antigen. Assay steps and assay time are indicated. IFCs were coated with commercial SARS-CoV-2 S-protein peptide and blocked with BSA. Corresponding antibody was diluted either in PBS or spiked in human serum and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized MNP. Finally, IFCs were inserted into the portable magnetic read-out device. Measuring signal can be correlated to the amount of antibody in the sample and antibody titer can be determined. Assay time of this preliminary MInD setup was 42 min which is approximately four times faster than ELISA (161 min).
Figure Legend Snippet: Proof-of-concept MInD assay setup using IFC coated with SARS-CoV-2 antigen. Assay steps and assay time are indicated. IFCs were coated with commercial SARS-CoV-2 S-protein peptide and blocked with BSA. Corresponding antibody was diluted either in PBS or spiked in human serum and applied to IFCs. Biotinylated secondary antibodies were added, followed by application of streptavidin-functionalized MNP. Finally, IFCs were inserted into the portable magnetic read-out device. Measuring signal can be correlated to the amount of antibody in the sample and antibody titer can be determined. Assay time of this preliminary MInD setup was 42 min which is approximately four times faster than ELISA (161 min).

Techniques Used: Antigen Assay, Enzyme-linked Immunosorbent Assay

ELISA-based detection of SARS-CoV-2 spike protein specific antibody in PBS-buffer (black curve) or spiked in human serum (red curve). Antibody was diluted to concentrations ranging from 1.22 ng·ml −1 up to 5000 ng·ml −1 in each matrix and applied onto 2 ng·ml −1 SARS-CoV-2 spike protein peptide coated and BSA blocked microtiter plates. After addition of biotinylated secondary antibody, streptavidin-AP was applied. Limit of detection (LOD) was calculated using non-linear Hill fit (R2=0.997 for PBS-buffer and 0.996 in serum). Assay time of ELISA was 161 minutes. Each data point represents mean ± SD (n = 4 for PBS-buffer and n = 3 for serum).
Figure Legend Snippet: ELISA-based detection of SARS-CoV-2 spike protein specific antibody in PBS-buffer (black curve) or spiked in human serum (red curve). Antibody was diluted to concentrations ranging from 1.22 ng·ml −1 up to 5000 ng·ml −1 in each matrix and applied onto 2 ng·ml −1 SARS-CoV-2 spike protein peptide coated and BSA blocked microtiter plates. After addition of biotinylated secondary antibody, streptavidin-AP was applied. Limit of detection (LOD) was calculated using non-linear Hill fit (R2=0.997 for PBS-buffer and 0.996 in serum). Assay time of ELISA was 161 minutes. Each data point represents mean ± SD (n = 4 for PBS-buffer and n = 3 for serum).

Techniques Used: Enzyme-linked Immunosorbent Assay, Serum Assay

29) Product Images from "Peripheral Blood T-Cell Fitness Is Diminished in Patients With Pancreatic Carcinoma but Can Be Improved With Homeostatic Cytokines"

Article Title: Peripheral Blood T-Cell Fitness Is Diminished in Patients With Pancreatic Carcinoma but Can Be Improved With Homeostatic Cytokines

Journal: Cellular and Molecular Gastroenterology and Hepatology

doi: 10.1016/j.jcmgh.2019.07.008

( A ) Cytokine secretion detected at 24 hours with T cells (patients [PT], n = 9) engineered to express a mesothelin-specific CAR and stimulated with mesothelin-expressing K562 cells. ( B ) Gene set enrichment analysis of T-cell effector signaling genes in peripheral blood CD8 + T cells isolated from patients ( n = 6) compared with healthy volunteers (HV) ( n = 6). ( C ) Heat map of genes expressed by CD3 + CD8 + T cells. ( D ) Messenger RNA levels of genes associated with differentiation, signaling, homing, and survival detected in sorted peripheral blood T cells as indicated. GM-CSF, granulocyte-macrophage–colony-stimulating factor. ∗ P
Figure Legend Snippet: ( A ) Cytokine secretion detected at 24 hours with T cells (patients [PT], n = 9) engineered to express a mesothelin-specific CAR and stimulated with mesothelin-expressing K562 cells. ( B ) Gene set enrichment analysis of T-cell effector signaling genes in peripheral blood CD8 + T cells isolated from patients ( n = 6) compared with healthy volunteers (HV) ( n = 6). ( C ) Heat map of genes expressed by CD3 + CD8 + T cells. ( D ) Messenger RNA levels of genes associated with differentiation, signaling, homing, and survival detected in sorted peripheral blood T cells as indicated. GM-CSF, granulocyte-macrophage–colony-stimulating factor. ∗ P

Techniques Used: Expressing, Isolation

30) Product Images from "The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells"

Article Title: The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells

Journal: Nature immunology

doi: 10.1038/ni.2912

Itch deficiency results in T cell–intrinsic defective differentiation of T FH cells. ( a ) Flow cytometry of activated (CD44 hi ) CD4 + T cells from wild-type (WT) and Itch −/− mice 8 d after infection with VACV. Numbers adjacent to outlined areas indicate percent CXCR5 + SLAM lo polyclonal T FH cells (top row) or CXCR5 + PD-1 hi GC T FH cells (middle row) or CXCR5 + Bcl-6 hi GC T FH cells (bottom row). ( b ) Frequency (among activated (CD44 hi ) CD4 + T cells) and total number of T FH cells and GC T FH cells in spleens of mice as in a ( n = 7 per group). ( c ) Flow cytometry of total B220 + B cells from mice as in a . Numbers adjacent to outlined areas indicate percent GL7 + Fas + GC B cells (top row) or CD138 + IgD lo plasma cells (bottom row). ( d ) Frequency (among total B220 + B cells) and total number of GC B cells and plasma cells in the spleen of mice as in a ( n = 7 per group). ( e ) ELISA of VACV-specific IgG in serum from infected mice as in a or uninfected B6 mice (Naive) ( n = 4 per group), presented as absorbance at 450 nm ( A 450 ). ( f ) Flow cytometry assessing polyclonal T FH cells and GC T FH cells from wild-type and Itch-cKO mice 8 d after infection with VACV (numbers in plots as in a ). ( g ) Frequency (among activated (CD44 hi ) CD4 + T cells) and total number of T FH cells and GC T FH cells in the spleen of mice as in f ( n = 6 per group). ( h ) Flow cytometry assessing GC B cells and plasma cells from mice as in f (numbers in plots as in c ). ( i ) Frequency (among total B220 + B cells) and total number of GC B cells and plasma cells in the spleen of mice as in f ( n = 6 per group). ( j ) ELISA of VACV-specific IgG in serum from mice as in f ( n = 3 per group). Each symbol ( b,d,g,i ) represents an individual mouse; small horizontal lines indicate the mean (±s.d.). * P
Figure Legend Snippet: Itch deficiency results in T cell–intrinsic defective differentiation of T FH cells. ( a ) Flow cytometry of activated (CD44 hi ) CD4 + T cells from wild-type (WT) and Itch −/− mice 8 d after infection with VACV. Numbers adjacent to outlined areas indicate percent CXCR5 + SLAM lo polyclonal T FH cells (top row) or CXCR5 + PD-1 hi GC T FH cells (middle row) or CXCR5 + Bcl-6 hi GC T FH cells (bottom row). ( b ) Frequency (among activated (CD44 hi ) CD4 + T cells) and total number of T FH cells and GC T FH cells in spleens of mice as in a ( n = 7 per group). ( c ) Flow cytometry of total B220 + B cells from mice as in a . Numbers adjacent to outlined areas indicate percent GL7 + Fas + GC B cells (top row) or CD138 + IgD lo plasma cells (bottom row). ( d ) Frequency (among total B220 + B cells) and total number of GC B cells and plasma cells in the spleen of mice as in a ( n = 7 per group). ( e ) ELISA of VACV-specific IgG in serum from infected mice as in a or uninfected B6 mice (Naive) ( n = 4 per group), presented as absorbance at 450 nm ( A 450 ). ( f ) Flow cytometry assessing polyclonal T FH cells and GC T FH cells from wild-type and Itch-cKO mice 8 d after infection with VACV (numbers in plots as in a ). ( g ) Frequency (among activated (CD44 hi ) CD4 + T cells) and total number of T FH cells and GC T FH cells in the spleen of mice as in f ( n = 6 per group). ( h ) Flow cytometry assessing GC B cells and plasma cells from mice as in f (numbers in plots as in c ). ( i ) Frequency (among total B220 + B cells) and total number of GC B cells and plasma cells in the spleen of mice as in f ( n = 6 per group). ( j ) ELISA of VACV-specific IgG in serum from mice as in f ( n = 3 per group). Each symbol ( b,d,g,i ) represents an individual mouse; small horizontal lines indicate the mean (±s.d.). * P

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

31) Product Images from "Preemptive donor apoptotic cell infusions induce IFN-γ-producing myeloid derived suppressor cells for cardiac allograft protection"

Article Title: Preemptive donor apoptotic cell infusions induce IFN-γ-producing myeloid derived suppressor cells for cardiac allograft protection

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302771

ECDI-SPs expanded Ly6C HI and Gr1 HI cells suppress CD8 + T cell proliferation
Figure Legend Snippet: ECDI-SPs expanded Ly6C HI and Gr1 HI cells suppress CD8 + T cell proliferation

Techniques Used:

Anti-Gr1 antibody depletes both Ly6C HI and Gr1 HI cells, concomitant with a significant increase in graft-infiltrating CD8 + T cells and graft loss
Figure Legend Snippet: Anti-Gr1 antibody depletes both Ly6C HI and Gr1 HI cells, concomitant with a significant increase in graft-infiltrating CD8 + T cells and graft loss

Techniques Used:

32) Product Images from "Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis"

Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109352

Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
Figure Legend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.
Figure Legend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

Techniques Used: Immunofluorescence, Staining, Cell Culture

33) Product Images from "Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus ▿"

Article Title: Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.05163-11

Immunolocalization of HEA. Immunolocalization of HEA was determined by Alexa Fluor 594-labeled goat anti-mouse secondary antibodies (red), and nuclear staining was identified by DAPI (blue). MAb 9B4 acted as an IgG1 isotype control antibody in this study.
Figure Legend Snippet: Immunolocalization of HEA. Immunolocalization of HEA was determined by Alexa Fluor 594-labeled goat anti-mouse secondary antibodies (red), and nuclear staining was identified by DAPI (blue). MAb 9B4 acted as an IgG1 isotype control antibody in this study.

Techniques Used: Labeling, Staining

34) Product Images from "Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer"

Article Title: Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0000000000000203

IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P
Figure Legend Snippet: IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P

Techniques Used: Expressing, Transduction, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P
Figure Legend Snippet: Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P

Techniques Used: Construct, Mouse Assay

5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P
Figure Legend Snippet: 5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P

Techniques Used: Construct, Transduction, Derivative Assay

Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.
Figure Legend Snippet: Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.

Techniques Used: Mouse Assay

35) Product Images from "Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid"

Article Title: Neuromyelitis optica study model based on chronic infusion of autoantibodies in rat cerebrospinal fluid

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-016-0577-8

Axonal damage and loss in the spinal cord and optic nerve of the NMO-rat. a Axon injury detected in the NMO-rat compared to the Control-rat (rats infused with IgG AQP4+ 2 and IgG Control 2, D7) using neurofilament immunostaining: reduced number of axons detected as NF-M-positive spots in the white matter (WM); fragmentation and reduced axon thickness in the gray matter (GM). b Classification (10–20 to 100–140 μm 2 , ImageJ) and quantification (mean by field) of NF-M-positive spots in the spinal cord of the NMO-rats ( n = 6) compared to the Control-rats ( n = 6): loss of axons with 60–140 μm 2 sections in the NMO-rats (in cart: evaluation of the total axon number, p = 0.03). c Co-detection of myelin alteration (MBP in green ) and axonal loss (neurofilament NF-M subtype in red ) in the spinal cord of the NMO-rat compared to the Control-rat. d Increased expression of the NF-H phosphorylated form, a marker of axon injury, detected by Western blot (pNF-H/NF-H ratio; p = 0.04). e Axon fragmentation and loss in the optic nerve of the NMO-rats compared to the Control-rat detected by NF-M immunostaining. Scale bars = 20 μm
Figure Legend Snippet: Axonal damage and loss in the spinal cord and optic nerve of the NMO-rat. a Axon injury detected in the NMO-rat compared to the Control-rat (rats infused with IgG AQP4+ 2 and IgG Control 2, D7) using neurofilament immunostaining: reduced number of axons detected as NF-M-positive spots in the white matter (WM); fragmentation and reduced axon thickness in the gray matter (GM). b Classification (10–20 to 100–140 μm 2 , ImageJ) and quantification (mean by field) of NF-M-positive spots in the spinal cord of the NMO-rats ( n = 6) compared to the Control-rats ( n = 6): loss of axons with 60–140 μm 2 sections in the NMO-rats (in cart: evaluation of the total axon number, p = 0.03). c Co-detection of myelin alteration (MBP in green ) and axonal loss (neurofilament NF-M subtype in red ) in the spinal cord of the NMO-rat compared to the Control-rat. d Increased expression of the NF-H phosphorylated form, a marker of axon injury, detected by Western blot (pNF-H/NF-H ratio; p = 0.04). e Axon fragmentation and loss in the optic nerve of the NMO-rats compared to the Control-rat detected by NF-M immunostaining. Scale bars = 20 μm

Techniques Used: Immunostaining, Expressing, Marker, Western Blot

36) Product Images from "The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation *"

Article Title: The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.357640

c-Cbl mediates Ag·BCR ubiquitination and processing. Upper panel , B cells were pulsed with anti-human IgM-btn for the indicated time (minutes) at 37 °C. The cells were lysed and ubiquitinated ligand-BCR complexes isolated by ubiquitin
Figure Legend Snippet: c-Cbl mediates Ag·BCR ubiquitination and processing. Upper panel , B cells were pulsed with anti-human IgM-btn for the indicated time (minutes) at 37 °C. The cells were lysed and ubiquitinated ligand-BCR complexes isolated by ubiquitin

Techniques Used: Isolation

37) Product Images from "The E3 ligase VHL promotes follicular helper T cell differentiation via glycolytic-epigenetic control"

Article Title: The E3 ligase VHL promotes follicular helper T cell differentiation via glycolytic-epigenetic control

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20190337

HIF-1α mediates VHL regulation of Tfh cell development. (A) Representative flow-cytometric analysis of CD44 + CD4 + T cells from WT, VHL cKO, and Vhl fl/fl Hif1a fl/fl CD4 Cre (DKO) mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 3, DKO group). (C) Representative flow-cytometric plots of total B220 + B cells from WT, VHL cKO, and DKO mice as in A. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ) cells (bottom row) in the spleen. (D) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 4, DKO group). (E) ELISA of LCMV-specific IgG in the sera from infected mice as in A ( n = 4 per group), presented as absorbance at 490 nm (A490). (F–H) RT-PCR analysis of quantification of LCMV copies in serum (F), spleen (G), and kidney (H) from WT, VHL cKO, and DKO mice 8 d after LCMV infection ( n = 3 or 4 per group). (I) Representative flow-cytometric plots of donor CD45.2 + CD4 + T cells obtained from WT CD45.1 + host mice receiving naive CD4 + T cells from WT, VHL cKO, and DKO SMARTA mice, followed by infection with LCMV and analysis 8 d after infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (J) Quantification of frequency (among CD45.2 + CD4 + T cells) and number (CD45.2 + CD4 + ) of Tfh cells and GC-Tfh cells of WT CD45.1 host mice as in I ( n = 3–5 per group). (K and L) Quantification of Bcl-6 geometric mean fluorescence intensity (gMFI) in WT or VHL cKO Tfh-like cells cultured in the absence or presence of px478 (K) or CoCl 2 (L). Each symbol (B, D, F–H, and J) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P
Figure Legend Snippet: HIF-1α mediates VHL regulation of Tfh cell development. (A) Representative flow-cytometric analysis of CD44 + CD4 + T cells from WT, VHL cKO, and Vhl fl/fl Hif1a fl/fl CD4 Cre (DKO) mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 3, DKO group). (C) Representative flow-cytometric plots of total B220 + B cells from WT, VHL cKO, and DKO mice as in A. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ) cells (bottom row) in the spleen. (D) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in A ( n = 7, WT group; n = 8, VHL cKO group; n = 4, DKO group). (E) ELISA of LCMV-specific IgG in the sera from infected mice as in A ( n = 4 per group), presented as absorbance at 490 nm (A490). (F–H) RT-PCR analysis of quantification of LCMV copies in serum (F), spleen (G), and kidney (H) from WT, VHL cKO, and DKO mice 8 d after LCMV infection ( n = 3 or 4 per group). (I) Representative flow-cytometric plots of donor CD45.2 + CD4 + T cells obtained from WT CD45.1 + host mice receiving naive CD4 + T cells from WT, VHL cKO, and DKO SMARTA mice, followed by infection with LCMV and analysis 8 d after infection. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (J) Quantification of frequency (among CD45.2 + CD4 + T cells) and number (CD45.2 + CD4 + ) of Tfh cells and GC-Tfh cells of WT CD45.1 host mice as in I ( n = 3–5 per group). (K and L) Quantification of Bcl-6 geometric mean fluorescence intensity (gMFI) in WT or VHL cKO Tfh-like cells cultured in the absence or presence of px478 (K) or CoCl 2 (L). Each symbol (B, D, F–H, and J) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P

Techniques Used: Flow Cytometry, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Cell Culture

VHL deficiency results in defective Tfh cell development and function. (A) Representative flow-cytometric plots of activated CD44 + CD4 + T cells from WT and CD4 Cre Vhl fl/fl (VHL cKO) mice 8 d after infection with LCMV. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 6 per group). (C) RT-PCR analysis of mRNA of Tfh cell–related genes in CD44 + CD4 + and CD44 − CD4 + T cells from WT and VHL cKO mice 8 d after LCMV infection; results were normalized to those of Actb mRNA (encoding β-actin). (D) Representative flow-cytometric plots of total B220 + B cells from WT and VHL cKO mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ; bottom row) cells in the spleen. (E) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in D ( n = 6 per group). (F) ELISA of LCMV-specific IgG in the sera from infected mice as in D ( n = 6 per group), presented as absorbance at 490 nm (A490). (G) Representative flow-cytometric plots of CD44 + CD4 + T cells from WT and VHL cKO mice 7 d after immunization with SRBC. Numbers adjacent to outlined areas indicate frequency of GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (H) Quantification of frequency (among CD44 + CD4 + T cells) and number of GC-Tfh cells of mice as in G ( n = 3–4 per group). Each symbol (B, E, and H) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P
Figure Legend Snippet: VHL deficiency results in defective Tfh cell development and function. (A) Representative flow-cytometric plots of activated CD44 + CD4 + T cells from WT and CD4 Cre Vhl fl/fl (VHL cKO) mice 8 d after infection with LCMV. Numbers adjacent to outlined areas indicate frequency of Tfh (CXCR5 + ICOS + or CXCR5 + SLAM lo ) cells or GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (B) Quantification of frequency (among CD44 + CD4 + T cells) and number of Tfh cells and GC-Tfh cells of mice as in A ( n = 6 per group). (C) RT-PCR analysis of mRNA of Tfh cell–related genes in CD44 + CD4 + and CD44 − CD4 + T cells from WT and VHL cKO mice 8 d after LCMV infection; results were normalized to those of Actb mRNA (encoding β-actin). (D) Representative flow-cytometric plots of total B220 + B cells from WT and VHL cKO mice 8 d after LCMV infection. Numbers adjacent to outlined areas indicate frequency of GC B (GL7 + CD95 + ; top row) or plasma (CD138 + IgD lo ; bottom row) cells in the spleen. (E) Quantification of frequency (among B220 + B cells) and number of GC B and plasma cells in the spleen of mice as in D ( n = 6 per group). (F) ELISA of LCMV-specific IgG in the sera from infected mice as in D ( n = 6 per group), presented as absorbance at 490 nm (A490). (G) Representative flow-cytometric plots of CD44 + CD4 + T cells from WT and VHL cKO mice 7 d after immunization with SRBC. Numbers adjacent to outlined areas indicate frequency of GC-Tfh (CXCR5 + PD-1 hi or CXCR5 + Bcl-6 hi ) cells. (H) Quantification of frequency (among CD44 + CD4 + T cells) and number of GC-Tfh cells of mice as in G ( n = 3–4 per group). Each symbol (B, E, and H) represents an individual mouse; small horizontal lines indicate the mean (± SD). *, P

Techniques Used: Flow Cytometry, Mouse Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

38) Product Images from "The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells"

Article Title: The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20160579

Th differentiation and B cell responses in mice deprived of functional RLTPR molecules. (A) Sorted naive CD4 + T cells (2 × 10 5 ) from mice of the specified genotype were stimulated for 5 d with anti-CD3 and -CD28 under Th1, Th2, or Th17 differentiating conditions. After 5 d of culture, the absolute number of IFN-γ + (Th1 condition), IL-4 + (Th2 condition), and IL-17 + (Th17 condition) CD4 + T cells was determined. Each dot corresponds to a mouse and the mean (horizontal bar) is indicated. (B) WT and Rltpr bas/bas mice were immunized intraperitoneally at day 0 and 14 with the T cell–dependent antigen TNP-KLH. The concentration of TNP-specific immunoglobulins of the indicated isotypes (IgG2a, IgG2b, and IgG1) were assessed in individual mice before and 21 d after immunization. (C) WT and Rltpr bas/bas mice were immunized with the T cell–independent antigen TNP-LPS, and the concentration of TNP-specific IgM was assessed in individual mice before and 7 d after immunization. (D) Splenic B cells from mice of the specified genotype were stimulated with F(ab)’ 2 goat anti–mouse IgM antibody in the presence or absence of anti-CD40 antibody, or LPS. After 4 d of culture, B cell proliferation was evaluated. Mean and SEM are shown. Data are representative of two independent experiments. In A–C, each dot corresponds to a mouse and the mean (horizontal bar) is indicated. **, P ≤ 0.01; ****, P ≤ 0.001; ns, nonsignificant. In D, two animals were used per genotype.
Figure Legend Snippet: Th differentiation and B cell responses in mice deprived of functional RLTPR molecules. (A) Sorted naive CD4 + T cells (2 × 10 5 ) from mice of the specified genotype were stimulated for 5 d with anti-CD3 and -CD28 under Th1, Th2, or Th17 differentiating conditions. After 5 d of culture, the absolute number of IFN-γ + (Th1 condition), IL-4 + (Th2 condition), and IL-17 + (Th17 condition) CD4 + T cells was determined. Each dot corresponds to a mouse and the mean (horizontal bar) is indicated. (B) WT and Rltpr bas/bas mice were immunized intraperitoneally at day 0 and 14 with the T cell–dependent antigen TNP-KLH. The concentration of TNP-specific immunoglobulins of the indicated isotypes (IgG2a, IgG2b, and IgG1) were assessed in individual mice before and 21 d after immunization. (C) WT and Rltpr bas/bas mice were immunized with the T cell–independent antigen TNP-LPS, and the concentration of TNP-specific IgM was assessed in individual mice before and 7 d after immunization. (D) Splenic B cells from mice of the specified genotype were stimulated with F(ab)’ 2 goat anti–mouse IgM antibody in the presence or absence of anti-CD40 antibody, or LPS. After 4 d of culture, B cell proliferation was evaluated. Mean and SEM are shown. Data are representative of two independent experiments. In A–C, each dot corresponds to a mouse and the mean (horizontal bar) is indicated. **, P ≤ 0.01; ****, P ≤ 0.001; ns, nonsignificant. In D, two animals were used per genotype.

Techniques Used: Mouse Assay, Functional Assay, Concentration Assay

39) Product Images from "T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *"

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1400663

Immunogenicity of purified VACV-WR antigen, measured by antibody production and protection against VACV-WR challenge in mice (A) IgG subtype analysis. Antibodies were engendered in C57Bl/6 mice against nickel column-purified VACV IMV membrane protein, WR101/H3LΔTM that has been adjuvanted in CpG/ISCOMs or alum, or in PBS alone as a control. Sera were obtained after 14 days and probed against VACV proteome microarrays on to which 8 two-fold serial dilutions of purified WR101/H3L were printed. Specific reactivity to purified WR101/H3L was visualized using fluorescently-tagged secondary antibodies to IgG, IgG1 and IgG2c and signal intensities quantified in a confocal laser scanner; data for a single concentration of printed antigen is shown. (B) Relative proportions of IgG1 and IgG2c derived from data shown in (A). The IgG2a proportion of the total signal is shown above the zero line, and the IgG1 proportion shown below. The IgG response is polarized according to adjuvant. (C) Protection of B6 mice against intranasal (i.n.) challenge of VACV-WR using adjuvanted WR101/H3LΔTM and WR101/H3L. CpG/ISCOMs reduce weight loss and promote recovery compared to alum or PBS. (D) and (E) correlations between nadir body weight (expressed as percentage of original body weight) and titer of IgG2c and IgG1, respectively. Titer was defined from the WR101/H3L titration series on the array at the lowest concentration to give a signal intensity > 2000. Liner regression was used to generate the trend lines.
Figure Legend Snippet: Immunogenicity of purified VACV-WR antigen, measured by antibody production and protection against VACV-WR challenge in mice (A) IgG subtype analysis. Antibodies were engendered in C57Bl/6 mice against nickel column-purified VACV IMV membrane protein, WR101/H3LΔTM that has been adjuvanted in CpG/ISCOMs or alum, or in PBS alone as a control. Sera were obtained after 14 days and probed against VACV proteome microarrays on to which 8 two-fold serial dilutions of purified WR101/H3L were printed. Specific reactivity to purified WR101/H3L was visualized using fluorescently-tagged secondary antibodies to IgG, IgG1 and IgG2c and signal intensities quantified in a confocal laser scanner; data for a single concentration of printed antigen is shown. (B) Relative proportions of IgG1 and IgG2c derived from data shown in (A). The IgG2a proportion of the total signal is shown above the zero line, and the IgG1 proportion shown below. The IgG response is polarized according to adjuvant. (C) Protection of B6 mice against intranasal (i.n.) challenge of VACV-WR using adjuvanted WR101/H3LΔTM and WR101/H3L. CpG/ISCOMs reduce weight loss and promote recovery compared to alum or PBS. (D) and (E) correlations between nadir body weight (expressed as percentage of original body weight) and titer of IgG2c and IgG1, respectively. Titer was defined from the WR101/H3L titration series on the array at the lowest concentration to give a signal intensity > 2000. Liner regression was used to generate the trend lines.

Techniques Used: Purification, Mouse Assay, Nickel Column, Concentration Assay, Derivative Assay, Titration

40) Product Images from "Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis"

Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109352

Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
Figure Legend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.
Figure Legend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

Techniques Used: Immunofluorescence, Staining, Cell Culture

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    Jackson Immuno biotin sp conjugated affinipure goat anti mouse
    Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated <t>AffiniPure</t> goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
    Biotin Sp Conjugated Affinipure Goat Anti Mouse, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno biotin sp affinipure goat anti mouse igg f ab 2 fragment specific
    Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated <t>AffiniPure</t> goat anti-mouse <t>IgG,F(ab′)2</t> fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
    Biotin Sp Affinipure Goat Anti Mouse Igg F Ab 2 Fragment Specific, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cytosolic <t>IgG/IgM</t> Ca 2+ responses and ER Ca 2+ content do not differ between Riva WT and Riva VR. ( a ) Typical cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT and VR. Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were stimulated with vehicle or 12 μg/mL IgG/IgM to provoke a cytosolic Ca 2+ signal. ( b ) Analysis of the area under the curve (AUC) of the IgG/IgM-triggered peak. Data are presented as average ± SEM (N = 5). ( c ) Cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT (black) and VR (red). Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were treated with thapsigargin (1 μM) to deplete ER Ca 2+ stores. Data are presented as average ± SEM (N = 6).
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    Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

    Journal: PLoS ONE

    Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

    doi: 10.1371/journal.pone.0109352

    Figure Lengend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

    Article Snippet: After washing, the cells were stained with biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment-specific antibody (Jackson ImmunoResearch 115-065-072), followed by PE-conjugated streptavidin (Jackson ImmunoResearch 016-110-084) and CD56-FITC.

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

    Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

    Journal: PLoS ONE

    Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

    doi: 10.1371/journal.pone.0109352

    Figure Lengend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

    Article Snippet: After washing, the cells were stained with biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment-specific antibody (Jackson ImmunoResearch 115-065-072), followed by PE-conjugated streptavidin (Jackson ImmunoResearch 016-110-084) and CD56-FITC.

    Techniques: Immunofluorescence, Staining, Cell Culture

    Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

    Journal: PLoS ONE

    Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

    doi: 10.1371/journal.pone.0109352

    Figure Lengend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

    Article Snippet: Biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment-specific antibody (Jackson ImmunoResearch 115-065-072) and PE-conjugated streptavidin (Jackson ImmunoResearch 016-110-084) were used for labeling the cells.

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

    Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

    Journal: PLoS ONE

    Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

    doi: 10.1371/journal.pone.0109352

    Figure Lengend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

    Article Snippet: Biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment-specific antibody (Jackson ImmunoResearch 115-065-072) and PE-conjugated streptavidin (Jackson ImmunoResearch 016-110-084) were used for labeling the cells.

    Techniques: Immunofluorescence, Staining, Cell Culture

    Cytosolic IgG/IgM Ca 2+ responses and ER Ca 2+ content do not differ between Riva WT and Riva VR. ( a ) Typical cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT and VR. Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were stimulated with vehicle or 12 μg/mL IgG/IgM to provoke a cytosolic Ca 2+ signal. ( b ) Analysis of the area under the curve (AUC) of the IgG/IgM-triggered peak. Data are presented as average ± SEM (N = 5). ( c ) Cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT (black) and VR (red). Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were treated with thapsigargin (1 μM) to deplete ER Ca 2+ stores. Data are presented as average ± SEM (N = 6).

    Journal: Biomolecules

    Article Title: DLBCL Cells with Acquired Resistance to Venetoclax Are Not Sensitized to BIRD-2 But Can Be Resensitized to Venetoclax through Bcl-XL Inhibition

    doi: 10.3390/biom10071081

    Figure Lengend Snippet: Cytosolic IgG/IgM Ca 2+ responses and ER Ca 2+ content do not differ between Riva WT and Riva VR. ( a ) Typical cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT and VR. Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were stimulated with vehicle or 12 μg/mL IgG/IgM to provoke a cytosolic Ca 2+ signal. ( b ) Analysis of the area under the curve (AUC) of the IgG/IgM-triggered peak. Data are presented as average ± SEM (N = 5). ( c ) Cytosolic Ca 2+ traces in Fura-2-AM-loaded Riva WT (black) and VR (red). Sixty seconds after the chelation of extracellular Ca 2+ with EGTA (3 mM), the cells were treated with thapsigargin (1 μM) to deplete ER Ca 2+ stores. Data are presented as average ± SEM (N = 6).

    Article Snippet: Extracellular Ca2+ was chelated with EGTA before stimulating cells with IgG/IgM (12 μg/mL; Jackson ImmunoResearch Europe Ltd., Cambridge, UK) to elicit intracellular Ca2+ signaling.

    Techniques: