mouse anti drd2  (Santa Cruz Biotechnology)

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: Leukemic progenitor assays replicate patterns of patient response to DRD2 antagonist TDZ (A) Leukemic blast counts were monitored before and after treatment with TDZ as a monotherapy in 11 relapsed or refractory AML patients (NCT02096289). Percentage change in blasts in the peripheral blood on day 5 versus day 1 is reported after treatment with TDZ. Percentage change in BM blast content is reported for trial patient 2T and 9T in the absence of circulating blast values. Partial response and progressive disease patterns are indicated as “response” and “no response” and are illustrated as gray versus black silhouettes, respectively. (B) Candidate trial patient samples from either response group were interrogated for progenitor content at baseline (day 1) and after clinical exposure to TDZ (day 5) using limiting dilution analysis (LDA). Leukemic progenitor frequency was estimated by LDA analysis and normalized to day 1. Baseline progenitor frequency of 1 in 75,000 cells was considered the progenitor frequency for trial patient 3T at day 1 since an absolute frequency was not achieved with the analysis of 75,000 cells for this patient. Dashed lines represent 95% confidence interval. Raw colony counts are shown in Figure S1 D. (C) Trial patient samples obtained at baseline were exposed to TDZ (“+TDZ”) versus DMSO control (“−TDZ”) for 24 h, followed by analysis of progenitor cell function in CFU assays. Data are normalized to DMSO control. Before normalization, the average DMSO control values were 79 and 2 colonies for trial patients 1T and 8T (non-responders) and 61, 28, 56, 2, 11, 28, and 14 colonies for trial patients 2T, 4T, 6T, 7T, 9T, 10T, and 11T, respectively (responders). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (D) Correlation between percentage change in leukemic blast levels versus percentage change in progenitor capacity (demonstrated in C). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (E) Schematic illustrating in vivo AML xenografts were treated with TDZ (22.5 mg/kg “+”) or 30% captisol (vehicle control “−”) in vivo , followed by analysis of leukemic chimerism levels (F), gene expression analysis (G), and progenitor CFU assays (H). (F) Leukemic chimerism levels (hCD45 + CD33 + ) after in vivo treatment with TDZ relative to vehicle control (“−“). Symbols represent individual recipient mice. ∗p = 0.05 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. (G) Gene set enrichment analysis (GSEA) plot of a gene set representing cellular pathways associated with AML (Kyoto Encyclopedia of Genes and Genomes [KEGG]; ), applied to transcription profiles from TDZ-treated versus vehicle control-treated AML xenografts derived from AMLs 1, 3, and 4. (H) Human AML grafts were recovered from mouse BM and evaluated in progenitor CFU assays. Symbols represent individual CFU wells, plated using cells recovered from a minimum of 2 individual mice per condition. Colony-forming capacity for AML 4 was not detectable with up to 150,000 human cells assayed. ∗∗∗p ≤ 0.0001 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. Data are summarized as means ± SEMs. See also and and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Control, Cell Function Assay, In Vivo, Gene Expression, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: DRD2 expression profiles reliably predict functional response to DRD antagonism (A) DRD2 expression patterns within leukemic CD34 + cells. Dotted line represents FMO control (left). Comparison of DRD2 protein levels in CD34 + cells of AML patient versus healthy donor samples (right). Healthy donor samples consist of cord blood (n = 3), adult mobilized peripheral blood (n = 3), and adult non-mobilized peripheral blood (n = 5). Blue versus red shading indicates the threshold of normal versus aberrant DRD2 levels. ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test). (B) DRD2 protein expression within CD34 + subset of low versus intermediate-/high-risk AML patients based on ELN criteria. Dots represent individual AML patients. ∗∗p = 0.006 (Mann-Whitney U test). (C) Mononuclear cells (MNCs) isolated from healthy donors and AML patients were treated with TDZ or DMSO (vehicle control, “−”) for 24 h and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n = 3–10 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). Source data can be found in . (D) Proliferative capacity of leukemic versus healthy progenitor units was compared after in vitro exposure to TDZ for 24 h. Cell number output per colony was evaluated by custom scripts as a measure of proliferation. (E) Representative FACS plots demonstrate gating strategy to purify DRD2 + vs DRD2 − human AML cells (left) and human leukemic chimerism in mice transplanted with 1 million DRD2 + or DRD2 − human AML cells. (F) Western blot of DRD2, activated CREB (p-CREB at Ser-133), and histone H3 (loading control) in DRD2 + versus DRD2 − sorted fractions illustrated in (E). (G) Representative whole-well CFU images after treatment with dopamine (DA) at physiological levels (10 nM) versus DMSO control (-DA). (H) Progenitor cell activity was quantified in n = 6 distinct AML patients after treatment with physiological levels of DA (10–100 nM) relative to DMSO control. n = 2–3 CFU wells per AML sample. ∗p = 0.03 (unpaired t test). (I) Circulating DA levels in healthy individuals (n = 8 healthy adult peripheral blood (PB) and 11 cord blood (CB) samples, as hollow circles and squares, respectively) versus n = 11 AML patients (black circles). ∗p = 0.04 (unpaired t test). Data are summarized as means ± SEMs relative to vehicle control. See also Figure S3 and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Expressing, Functional Assay, Control, Comparison, MANN-WHITNEY, Isolation, In Vitro, Western Blot, Activity Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: cAMP elevation is associated with leukemic progenitor suppression (A) Trial patients (NCT02096289) were exposed to TDZ in vitro , followed by analysis of cAMP level changes. Trial patients with abundant cell numbers available were prioritized for this analysis, including patients 1T and 3T from non-responders, and patients 7T, 10T, and 11T for responders. n = 3–6 technical replicates per condition. ∗p ≤ 0.05 (unpaired t test). (B) cAMP levels in response to DRD1 agonist (SKF 38393) relative to DMSO control. n ≥ 4 replicates across OCI-AML3 and NB4 cell lines. ∗∗p = 0.008 (Mann-Whitney U test). Progenitor response was evaluated after treatment with DRD1 agonist (SKF 38393) relative to DMSO control. n = 2–3 CFU replicates per AML sample (n = 5 AML samples total). (C) cAMP levels in response to anti-DRD1 antibody alone or in combination with DRD1 antagonist (SCH 23390) in AML cell lines OCI-AML3 and NB4. n = 2–4 replicates per condition. (D) Western blot of activated CREB (p-CREB at Ser-133) after exposure to anti-DRD1 antibody in OCI-AML3 cell line (top). Western blot of activated CREB (p-CREB at Ser-133) exposure to TDZ in OCI-AML3 and NB4 cell lines (bottom). (E) MNCs isolated from healthy donors and AML patients were treated with anti-DRD1 antibody or immunoglobulin G (IgG) control (“−“) for 30 min, and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n =3–7 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). (F) Cytospin preparations of AML cells from patient 2 after exposure to TDZ or vehicle control (DMSO). Yellow arrowheads indicate evidence of hematopoietic maturation (increased cell size, reduced nuclear:cytoplasmic ratio, increased cytoplasmic vacuolization). (G) FACS plot showing expression of granulocytic cell marker (CD15) after in vitro exposure to TDZ or DMSO control (“-TDZ“) in representative DRD2 lo and DRD2 + AML samples. CD15 frequencies were quantified for AMLs 1, 6, and 7 (n = 2 technical replicates per AML sample in each condition). ∗∗p = 0.002 (Mann-Whitney U test). (H) AML patient cells were treated with TDZ or DMSO for 24 h and evaluated in progenitor CFU assays, followed by analysis of re-plating capacity. ∗∗p = 0.004 (unpaired t test). (I) cAMP levels in response to TDZ relative to DMSO control. DRD2 + AML includes AML 1, 6, OCI-AML3, and NB4. DRD2 − AML and healthy controls include AML 12 and 3 CB samples, respectively. n ≥ 3 replicates per condition. ∗∗∗p = 0.007 (unpaired t test). (J) cAMP levels in response to forskolin (FSK) relative to DMSO control. n = 6 replicates per condition, across 1 AML cell line and n = 2 healthy donor cells. ∗∗∗p ≤ 0.0001 (unpaired t test). Data are summarized as means ± SEMs. See also Figure S4 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: In Vitro, Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: TDZ + displays superior potency and reduced toxicity relative to TDZ (A) Chiral separation of TDZ using supercritical fluid chromatography. Chromatograms show the first and second peaks, indicating the (−) enantiomer “TDZ − ” and (+) enantiomer “TDZ + ,” respectively. Purified enantiomers were evaluated for effects on cAMP levels (B), and in progenitor CFU assays (C and D). (B) cAMP levels were evaluated after in vitro treatment with TDZ and its two enantiomers in AML cell lines (NB4 and OCI-AML3) and primary patient cells (AMLs 2, 9, and 27). Symbols represent individual CFU wells. ∗p ≤ 0.05 and ∗∗p ≤ 0.01 (unpaired t test). (C) AML patient cells were exposed to TDZ and its 2 enantiomers for 24 h in a dose-response assay in vitro , and subsequently evaluated in progenitor CFU assays. Bar graphs summarize half-maximal inhibitory concentration (IC 50 ) in progenitor CFU assays performed with AML patient cells. ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001 (paired t test). (D) Comparison of TDZ and TDZ + IC 50 for individual AML patients in CFU assays (represented in C). ∗∗p = 0.004 (paired t test). (E) A 30-min monitoring of QTc level changes after intravenous injection of TDZ and TDZ + in a guinea pig assay (n = 5 animals per cohort). QTc increases over 5% were considered indicators of safety risks. No group averages were statistically different from baseline values (repeated-measures ANOVAs). (F) DRD2 transcript (Gene: 1813) was analyzed from TGCA (tumor and normal tissue) and GTEx (normal tissue) RNA-sequencing projects. Data points represent normalized gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) for DRD2 from individual cancer patients or healthy donors. ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test), ∗∗p = 0.01 (Kolmogorov-Smirnov test). Data are summarized as means ± SEMs. See also Figure S5 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Supercritical Fluid Chromatography, Purification, In Vitro, Concentration Assay, Comparison, Injection, RNA Sequencing, Gene Expression, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet:

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Recombinant, Binding Assay, Purification, Microarray, Software, Imaging

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: Leukemic progenitor assays replicate patterns of patient response to DRD2 antagonist TDZ (A) Leukemic blast counts were monitored before and after treatment with TDZ as a monotherapy in 11 relapsed or refractory AML patients (NCT02096289). Percentage change in blasts in the peripheral blood on day 5 versus day 1 is reported after treatment with TDZ. Percentage change in BM blast content is reported for trial patient 2T and 9T in the absence of circulating blast values. Partial response and progressive disease patterns are indicated as “response” and “no response” and are illustrated as gray versus black silhouettes, respectively. (B) Candidate trial patient samples from either response group were interrogated for progenitor content at baseline (day 1) and after clinical exposure to TDZ (day 5) using limiting dilution analysis (LDA). Leukemic progenitor frequency was estimated by LDA analysis and normalized to day 1. Baseline progenitor frequency of 1 in 75,000 cells was considered the progenitor frequency for trial patient 3T at day 1 since an absolute frequency was not achieved with the analysis of 75,000 cells for this patient. Dashed lines represent 95% confidence interval. Raw colony counts are shown in Figure S1 D. (C) Trial patient samples obtained at baseline were exposed to TDZ (“+TDZ”) versus DMSO control (“−TDZ”) for 24 h, followed by analysis of progenitor cell function in CFU assays. Data are normalized to DMSO control. Before normalization, the average DMSO control values were 79 and 2 colonies for trial patients 1T and 8T (non-responders) and 61, 28, 56, 2, 11, 28, and 14 colonies for trial patients 2T, 4T, 6T, 7T, 9T, 10T, and 11T, respectively (responders). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (D) Correlation between percentage change in leukemic blast levels versus percentage change in progenitor capacity (demonstrated in C). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (E) Schematic illustrating in vivo AML xenografts were treated with TDZ (22.5 mg/kg “+”) or 30% captisol (vehicle control “−”) in vivo , followed by analysis of leukemic chimerism levels (F), gene expression analysis (G), and progenitor CFU assays (H). (F) Leukemic chimerism levels (hCD45 + CD33 + ) after in vivo treatment with TDZ relative to vehicle control (“−“). Symbols represent individual recipient mice. ∗p = 0.05 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. (G) Gene set enrichment analysis (GSEA) plot of a gene set representing cellular pathways associated with AML (Kyoto Encyclopedia of Genes and Genomes [KEGG]; ), applied to transcription profiles from TDZ-treated versus vehicle control-treated AML xenografts derived from AMLs 1, 3, and 4. (H) Human AML grafts were recovered from mouse BM and evaluated in progenitor CFU assays. Symbols represent individual CFU wells, plated using cells recovered from a minimum of 2 individual mice per condition. Colony-forming capacity for AML 4 was not detectable with up to 150,000 human cells assayed. ∗∗∗p ≤ 0.0001 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. Data are summarized as means ± SEMs. See also and and .

Article Snippet: Immunophenotyping of cell surface markers was carried out using CD45 (642275, clone 2D1, BD PharMingen; 564048, clone H130, BD Horizon), CD34 (555822, clone 581, BD PharMingen; 555824, 581, BD Biosciences), CD33 (551378, clone WM53, BD PharMingen; 565949, clone WM53, BD Horizon), CD15 (555401, clone HI98, BD Biosciences; IM1954U, clone 80H5, Beckman Coulter), Rabbit anti-human DRD1 antibody (324390, EMD Millipore) and Mouse anti-human DRD2 antibody (clone B-10, Santa Cruz).

Techniques: Control, Cell Function Assay, In Vivo, Gene Expression, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: DRD2 expression profiles reliably predict functional response to DRD antagonism (A) DRD2 expression patterns within leukemic CD34 + cells. Dotted line represents FMO control (left). Comparison of DRD2 protein levels in CD34 + cells of AML patient versus healthy donor samples (right). Healthy donor samples consist of cord blood (n = 3), adult mobilized peripheral blood (n = 3), and adult non-mobilized peripheral blood (n = 5). Blue versus red shading indicates the threshold of normal versus aberrant DRD2 levels. ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test). (B) DRD2 protein expression within CD34 + subset of low versus intermediate-/high-risk AML patients based on ELN criteria. Dots represent individual AML patients. ∗∗p = 0.006 (Mann-Whitney U test). (C) Mononuclear cells (MNCs) isolated from healthy donors and AML patients were treated with TDZ or DMSO (vehicle control, “−”) for 24 h and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n = 3–10 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). Source data can be found in . (D) Proliferative capacity of leukemic versus healthy progenitor units was compared after in vitro exposure to TDZ for 24 h. Cell number output per colony was evaluated by custom scripts as a measure of proliferation. (E) Representative FACS plots demonstrate gating strategy to purify DRD2 + vs DRD2 − human AML cells (left) and human leukemic chimerism in mice transplanted with 1 million DRD2 + or DRD2 − human AML cells. (F) Western blot of DRD2, activated CREB (p-CREB at Ser-133), and histone H3 (loading control) in DRD2 + versus DRD2 − sorted fractions illustrated in (E). (G) Representative whole-well CFU images after treatment with dopamine (DA) at physiological levels (10 nM) versus DMSO control (-DA). (H) Progenitor cell activity was quantified in n = 6 distinct AML patients after treatment with physiological levels of DA (10–100 nM) relative to DMSO control. n = 2–3 CFU wells per AML sample. ∗p = 0.03 (unpaired t test). (I) Circulating DA levels in healthy individuals (n = 8 healthy adult peripheral blood (PB) and 11 cord blood (CB) samples, as hollow circles and squares, respectively) versus n = 11 AML patients (black circles). ∗p = 0.04 (unpaired t test). Data are summarized as means ± SEMs relative to vehicle control. See also Figure S3 and .

Article Snippet: Immunophenotyping of cell surface markers was carried out using CD45 (642275, clone 2D1, BD PharMingen; 564048, clone H130, BD Horizon), CD34 (555822, clone 581, BD PharMingen; 555824, 581, BD Biosciences), CD33 (551378, clone WM53, BD PharMingen; 565949, clone WM53, BD Horizon), CD15 (555401, clone HI98, BD Biosciences; IM1954U, clone 80H5, Beckman Coulter), Rabbit anti-human DRD1 antibody (324390, EMD Millipore) and Mouse anti-human DRD2 antibody (clone B-10, Santa Cruz).

Techniques: Expressing, Functional Assay, Control, Comparison, MANN-WHITNEY, Isolation, In Vitro, Western Blot, Activity Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: cAMP elevation is associated with leukemic progenitor suppression (A) Trial patients (NCT02096289) were exposed to TDZ in vitro , followed by analysis of cAMP level changes. Trial patients with abundant cell numbers available were prioritized for this analysis, including patients 1T and 3T from non-responders, and patients 7T, 10T, and 11T for responders. n = 3–6 technical replicates per condition. ∗p ≤ 0.05 (unpaired t test). (B) cAMP levels in response to DRD1 agonist (SKF 38393) relative to DMSO control. n ≥ 4 replicates across OCI-AML3 and NB4 cell lines. ∗∗p = 0.008 (Mann-Whitney U test). Progenitor response was evaluated after treatment with DRD1 agonist (SKF 38393) relative to DMSO control. n = 2–3 CFU replicates per AML sample (n = 5 AML samples total). (C) cAMP levels in response to anti-DRD1 antibody alone or in combination with DRD1 antagonist (SCH 23390) in AML cell lines OCI-AML3 and NB4. n = 2–4 replicates per condition. (D) Western blot of activated CREB (p-CREB at Ser-133) after exposure to anti-DRD1 antibody in OCI-AML3 cell line (top). Western blot of activated CREB (p-CREB at Ser-133) exposure to TDZ in OCI-AML3 and NB4 cell lines (bottom). (E) MNCs isolated from healthy donors and AML patients were treated with anti-DRD1 antibody or immunoglobulin G (IgG) control (“−“) for 30 min, and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n =3–7 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). (F) Cytospin preparations of AML cells from patient 2 after exposure to TDZ or vehicle control (DMSO). Yellow arrowheads indicate evidence of hematopoietic maturation (increased cell size, reduced nuclear:cytoplasmic ratio, increased cytoplasmic vacuolization). (G) FACS plot showing expression of granulocytic cell marker (CD15) after in vitro exposure to TDZ or DMSO control (“-TDZ“) in representative DRD2 lo and DRD2 + AML samples. CD15 frequencies were quantified for AMLs 1, 6, and 7 (n = 2 technical replicates per AML sample in each condition). ∗∗p = 0.002 (Mann-Whitney U test). (H) AML patient cells were treated with TDZ or DMSO for 24 h and evaluated in progenitor CFU assays, followed by analysis of re-plating capacity. ∗∗p = 0.004 (unpaired t test). (I) cAMP levels in response to TDZ relative to DMSO control. DRD2 + AML includes AML 1, 6, OCI-AML3, and NB4. DRD2 − AML and healthy controls include AML 12 and 3 CB samples, respectively. n ≥ 3 replicates per condition. ∗∗∗p = 0.007 (unpaired t test). (J) cAMP levels in response to forskolin (FSK) relative to DMSO control. n = 6 replicates per condition, across 1 AML cell line and n = 2 healthy donor cells. ∗∗∗p ≤ 0.0001 (unpaired t test). Data are summarized as means ± SEMs. See also Figure S4 .

Article Snippet: Immunophenotyping of cell surface markers was carried out using CD45 (642275, clone 2D1, BD PharMingen; 564048, clone H130, BD Horizon), CD34 (555822, clone 581, BD PharMingen; 555824, 581, BD Biosciences), CD33 (551378, clone WM53, BD PharMingen; 565949, clone WM53, BD Horizon), CD15 (555401, clone HI98, BD Biosciences; IM1954U, clone 80H5, Beckman Coulter), Rabbit anti-human DRD1 antibody (324390, EMD Millipore) and Mouse anti-human DRD2 antibody (clone B-10, Santa Cruz).

Techniques: In Vitro, Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: TDZ + displays superior potency and reduced toxicity relative to TDZ (A) Chiral separation of TDZ using supercritical fluid chromatography. Chromatograms show the first and second peaks, indicating the (−) enantiomer “TDZ − ” and (+) enantiomer “TDZ + ,” respectively. Purified enantiomers were evaluated for effects on cAMP levels (B), and in progenitor CFU assays (C and D). (B) cAMP levels were evaluated after in vitro treatment with TDZ and its two enantiomers in AML cell lines (NB4 and OCI-AML3) and primary patient cells (AMLs 2, 9, and 27). Symbols represent individual CFU wells. ∗p ≤ 0.05 and ∗∗p ≤ 0.01 (unpaired t test). (C) AML patient cells were exposed to TDZ and its 2 enantiomers for 24 h in a dose-response assay in vitro , and subsequently evaluated in progenitor CFU assays. Bar graphs summarize half-maximal inhibitory concentration (IC 50 ) in progenitor CFU assays performed with AML patient cells. ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001 (paired t test). (D) Comparison of TDZ and TDZ + IC 50 for individual AML patients in CFU assays (represented in C). ∗∗p = 0.004 (paired t test). (E) A 30-min monitoring of QTc level changes after intravenous injection of TDZ and TDZ + in a guinea pig assay (n = 5 animals per cohort). QTc increases over 5% were considered indicators of safety risks. No group averages were statistically different from baseline values (repeated-measures ANOVAs). (F) DRD2 transcript (Gene: 1813) was analyzed from TGCA (tumor and normal tissue) and GTEx (normal tissue) RNA-sequencing projects. Data points represent normalized gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) for DRD2 from individual cancer patients or healthy donors. ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test), ∗∗p = 0.01 (Kolmogorov-Smirnov test). Data are summarized as means ± SEMs. See also Figure S5 .

Article Snippet: Immunophenotyping of cell surface markers was carried out using CD45 (642275, clone 2D1, BD PharMingen; 564048, clone H130, BD Horizon), CD34 (555822, clone 581, BD PharMingen; 555824, 581, BD Biosciences), CD33 (551378, clone WM53, BD PharMingen; 565949, clone WM53, BD Horizon), CD15 (555401, clone HI98, BD Biosciences; IM1954U, clone 80H5, Beckman Coulter), Rabbit anti-human DRD1 antibody (324390, EMD Millipore) and Mouse anti-human DRD2 antibody (clone B-10, Santa Cruz).

Techniques: Supercritical Fluid Chromatography, Purification, In Vitro, Concentration Assay, Comparison, Injection, RNA Sequencing, Gene Expression, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet:

Article Snippet: Immunophenotyping of cell surface markers was carried out using CD45 (642275, clone 2D1, BD PharMingen; 564048, clone H130, BD Horizon), CD34 (555822, clone 581, BD PharMingen; 555824, 581, BD Biosciences), CD33 (551378, clone WM53, BD PharMingen; 565949, clone WM53, BD Horizon), CD15 (555401, clone HI98, BD Biosciences; IM1954U, clone 80H5, Beckman Coulter), Rabbit anti-human DRD1 antibody (324390, EMD Millipore) and Mouse anti-human DRD2 antibody (clone B-10, Santa Cruz).

Techniques: Recombinant, Binding Assay, Purification, Microarray, Software, Imaging

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 1 Association of CHL1 and DRD2. Brain extracts from CHL1+/+ (A-C) or CHL1−/− (C) mice (input) were subjected to immunoprecipitation (IP) with anti-CHL1 (A, C), anti-NCAM (A), anti-DRD2 (B), or non-immune control antibodies (A, B) and to Western blot analysis (WB) with anti-DRD2 (A, C) or anti-CHL1 (B) antibodies. D, CHL1-Fc and NCAM-Fc were incubated with a brain extract from CHL1+/+ mice (input) followed by pull-down (PD) with Protein A beads and by Western blot analysis (WB) with anti-DRD2 antibody. A-D, Representative Western blots from three independent experiments are shown. Lanes not adjacent to each other but derived from the same blot are separated by a vertical line. Aliquots of brain extracts not subjected to immunoprecipitation or pull-down experiments are designated as input

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Immunoprecipitation, Control, Western Blot, Incubation, Derivative Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 2 Binding of CHL1-Fc to the first extracellular loop of DRD2. Synthetic peptides comprising the first (A), second (B), or third (C) extracellular DRD2 loop or the DRD2 N-terminus (D) were coated as substrate and incubated with increasing concentrations of CHL1-Fc or NCAM-Fc. Binding was determined by ELISA using horseradish peroxidase-conjugated anti-Fc antibodies. Mean values ± standard error of the mean from three independent experiments carried out in triplicates are shown

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 3 Binding of CHL1-Fc to DRD2 at the cell surface of transfected HEK293 cells. Live HEK293 cell expressing DRD2-S or DRD2-L were incubated with CHL1-Fc. After fixation, cells were incubated with goat anti-CHL1 antibody and mouse anti-DRD2 antibody against the extracellular N-terminus. After washing, cells were stained with Cy2-conjugated anti-mouse (green) and Cy3- conjugated anti-goat (red) antibodies. Areas indicated by white dashed lines and arrows show areas with CHL1-immunopositive signals, which partially overlap with intense DRD2-immunopositive staining. Arrowheads indicate nuclei of CHL1- and DRD2-immunonegative untransfected cells. The experiment was performed two times. Scale bar: 10 µm

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Binding Assay, Transfection, Expressing, Incubation, Staining

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 4 Interaction of CHL1 with DRD2-S and DRD2-L in transfected HEK293 cells. HEK293 cells were transfected with pCAG- DRD2-S, pCAG-DRD2-L, pCAG-DRD2-S/CHL1, or pCAG-DRD2-L/CHL1 to express DRD2-S or DRD2-L alone or together with CHL1 (DRD2-S/CHL1; DRD2-L/CHL1). Transfected cells were subjected to proximity ligation assay with anti-CHL1 and anti-DRD2 antibodies. A, Representative images at low (left and middle panel) and high magnification (right panels) from two independent experiments performed in duplicates are shown and red spots indicate close interaction of CHL1 with DRD2-S and DRD2-L. Scale bars: 10 µm. B, Red spots were counted in HEK293 cells co-expressing DRD2-S or DRD2-L and CHL1 (DRD2-S/CHL1; DRD2-L/CHL1). Numbers of red spots per cell from two independent experiments performed in duplicates are shown (*P < .001; two-tailed Student's t test). C, Representative images taken with and without phase contrast are shown. Red spots are located predominantly at the cell surface (arrows) and indicate the close interaction between CHL1 and DRD2-S at the cell surface

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Transfection, Proximity Ligation Assay, Expressing, Two Tailed Test

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 5 Quinpirole reduces the cell surface level of DRD2-S in the absence of CHL1. A-C, HEK293 expressing DRD2-S (A) or DRD2-L (B) alone or co-expressing DRD2-S and CHL1 (DRD2-S/CHL1) (A, C) or DRD2-L and CHL1 (DRD2-L/CHL1) (B, C) were incubated with (+quin) or without (−quin) quinpirole followed by cell surface biotinylation, isolation of biotinylated proteins and Western blot analysis (WB) of the biotinylated proteins (surface) and the cell lysates (total) with anti-DRD2 (A, B) and anti-CHL1 (C) antibodies. The anti-GAPDH antibody was used to control loading (A, B). Total levels indicate the levels of CHL1 and DRD2 in cell lysates before isolation of biotinylated proteins, and cell surface levels represent biotinylated DRD2 after isolation of biotinylated proteins. A-C, Shown are representative blots from three independent experiments. D, E, Cell surface levels of DRD2-S and DRD2-L and total DRD2-S and DRD2-L levels in the cell lysates were determined, cell surface levels were normalized to total levels and the ratio of relative cell surface level after quinpirole treatment (+quin) and relative cell surface level after treatment without quinpirole (−quin) was calculated. Means + standard deviation from three independent experiments are shown for the ratios of the cell surface levels with quinpirole treatment relative to the cell surface levels without quinpirole treatment (D) and for the cell surface levels without quinpirole treatment relative to the total levels without quinpirole treatment (E) (Kruskal-Wallis test with post-hoc Dunn´s multiple comparison test; **P < .01; ns: not significant)

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Incubation, Isolation, Western Blot, Control, Standard Deviation, Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 6 Quinpirole-induced internalization of DRD2-S is reduced in the presence of CHL1. A, B, HEK293 expressing DRD2-S or co- expressing DRD2-S and CHL1 (DRD2-S/CHL1) were incubated with anti-DRD2 antibody against the extracellular N-terminus. After removal of unbound antibodies, cells were stimulated without (−quin) or with (+quin) quinpirole, fixed and incubated with Cy-3-conjugated secondary antibody. After removal of unbound secondary antibodies, cells were permeabilized and incubated with Cy-2-conjugated secondary antibody and analyzed by confocal microscopy. Cy-3-conjugated secondary antibodies (red) indicate non-internalized DRD2-bound anti-DRD2 antibodies at the cell surface and Cy-2-conjugated secondary antibodies (green) label internalized DRD2-bound anti-DRD2 antibodies. A, Representative images of HEK293 expressing DRD2-S after quinpirole treatment show surface DRD2 (red) and internalized DRD2 (green). Scale bar: 10 µm. B, Integrated densities of internalized and cell surface receptor-bound antibodies were determined and integrated densities of internalized receptor-bound antibodies were normalized to the total integrated densities (sum of integrated densities of internalized and cell surface receptor-bound antibodies). Box plots for the relative levels of internalized DRD2 are shown (**P < .01, ***P < .001; One-way ANOVA with post-hoc Student Newman- Keul's test). The experiment was performed two times in duplicates

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Incubation, Confocal Microscopy, Cell Surface Receptor Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 7 Co-immunostaining of CHL1 and DRD2 in the striatum. Tissue sections from 12- to 18-week-old CHL1+/+ (A, C) and CHL1−/− (B) mice were subjected to immunostaining using goat anti-CHL1 and mouse anti-DRD2 antibodies and Cy-3-conjugated anti- mouse and Cy-2-conjugated anti-goat secondary antibodies. Nuclei are stained with DAPI. Representative image of immunofluorescence staining for DAPI (blue), CHL1 (green), and DRD2 (red) are shown and yellow signals show co- localizations. C, Close-ups of two regions indicated by boxes in (A). A-C, Scale bars: 20 µm. Three independent experiments were performed with different sets of animals

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Immunostaining, Staining, Immunofluorescence

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 8 Co-localization of CHL1 and DRD2 in the striatum. Tissue sections from 12- to 18-week-old CHL1+/+ and CHL1−/− mice were analyzed by proximity ligation assay using goat anti-CHL1 and mouse anti-DRD2 antibodies. Nuclei are stained with DAPI (blue). Representative images are shown at low (left and middle panel) and high (right panel) magnifications. Red spots indicate close molecular interaction of CHL1 with DRD2 in CHL1+/+ mice. CHL1−/− mice served as controls. Scale bars: 10 µm. Three independent experiments were performed with different sets of animals

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Proximity Ligation Assay, Staining

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 9 Interaction of CHL1 and DRD2 on TH- and DARP32-positive neurons in striatal sections. Proximity ligation assay using goat anti-CHL1 and mouse anti-DRD2 antibodies was combined with immunostaining using rabbit anti-DARPP32 (A) or anti-TH (B) antibodies to analyze tissue sections from 12- to 18-week-old CHL1+/+ mice. Nuclei are stained with DAPI (blue). Representative images are shown. Close- ups of two regions (without DAPI staining) are indicated by boxes and arrowheads indicate red spots indicating close molecular interaction of CHL1 with DRD2. Scale bars: 10 µm. Three independent experiments were performed with different sets of animals

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Proximity Ligation Assay, Immunostaining, Staining

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 10 Interaction of CHL1 and DRD2 on TH- and DARP32-positive cells in cultures of ventral midbrain and striatum. Cultures of ventral midbrain (A) or striatum (B) were analyzed by proximity ligation assay using goat anti-CHL1 and mouse anti-DRD2 antibodies combined with immunofluorescent staining using rabbit anti-TH or anti-DARPP32 antibodies. Nuclei are stained with DAPI (blue). Representative images of different cells are shown. Red spots indicate close molecular interaction between CHL1 and DRD2. Scale bars: 10 µm. Three independent experiments were performed with different sets of animals

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Proximity Ligation Assay, Staining

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

doi: 10.1096/fj.201900577RRRR

Figure Lengend Snippet: FIGURE 11 Reduced DRD2 and pSer40-TH levels in the dorsal striatum and reduced pThr34-DARPP32 levels in the ventral striatum in the absence of CHL1. The dorsal and ventral parts of the striatum were isolated from 10- to 13-week-old CHL1+/+ and CHL1−/− mice and subjected to Western blot analysis with anti-DRD2 and anti-GAPDH antibodies (A), anti-pSer40-TH and anti-TH antibodies (B) or anti-pThr34-DARPP32 and anti-DARPP32 (C) antibodies. Protein levels were determined by densitometry and DRD2 levels relative to GAPDH levels (A), pSer40-TH levels relative to total TH levels (B) and pThr34-DARPP32 levels relative to total DARPP32 levels (C) were calculated. A-C, Representative Western blots (left panels) are shown and mean values + standard error of the mean from eight CHL1−/− and 11 CHL1+/+ mice (right panels) are shown for the relative levels of DRD2 (A), pSer40-TH (B) and pThr34-DARPP32 (C) (Kruskal-Wallis test with post-hoc Dunn´s multiple comparison test; *P < .05, **P < .01). A, Lanes not adjacent to each other but derived from the same blot are separated by a vertical line

Article Snippet: The mouse monoclonal antibody against DRD2 (Santa Cruz Biotechnology Cat# sc-5303, RRID:AB_668816), the rabbit polyclonal antibody against GAPDH (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668), and Protein A/G agarose beads were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Isolation, Western Blot, Comparison, Derivative Assay