Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: IL‐25R is highly expressed by in vitro differentiated CRTh2 + Th2 cells. Flow cytometry of (A) surface CRTh2 (solid line) compared with isotype control (dotted line; day 42) and (B) intra‐cellular IL‐4, IL‐13 and IFN‐γ⋅ (C) qRT‐PCR for IL‐25R mRNA from activated CRTh2 + Th2 cells (day 45, black bar, n = 6 independently differentiated lines) were compared with activated non‐polarized CD4 + T cells (day 3, white bar, 61‐fold, n = 5). (D) Representative example of surface IL‐25R expression (solid line) compared to isotype control (dotted line) by differentiated CRTh2 + Th2 cells (day 43) and (E) kinetics of surface IL‐25R expression following TCR activation ( n = 4, three independently differentiated lines). Statistical significance was determined by Student's t test, * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: In Vitro, Flow Cytometry, Control, Quantitative RT-PCR, Expressing, Activation Assay

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: IL‐25 mediates acquisition of the Th2 phenotype. Naïve CD4 + T cells were differentiated in non‐polarizing (NP) control conditions (αCD3/αCD28, IL‐2, αIFNγ and αIL‐12) or NP + IL‐4 (IL‐4), NP + IL‐25 (IL‐25) or NP + IL‐4 and IL‐25 (IL‐4+IL‐25). (A) IL‐25R mRNA expression (fold increase over NP; day 3, 7, 10, 14, n = 10 of three independently differentiated lines). (B) Intracellular IL‐4 and (E) surface CRTh2 were assayed following proliferation (day 7, 14, n = 17, 10 independently differentiated lines). (D) GATA3 was assayed after stimulation (day 3, 10, n = 17, 10 independently differentiated lines). (C) IL‐4 mRNA expression by cells cultured in IL‐4 or IL‐25 (fold increase over NP, day 14; n = 4 independently differentiated lines). (F) Cell counts were determined and comparison was made between the four conditions for each day. Statistical significance determined by repeated measures ANOVA, * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: Control, Expressing, Cell Culture, Comparison

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: Regulation of IL‐25R on Th2 cells. Differentiated CRTh2 + Th2 cells were cultured with IL‐2 in the presence or absence of IL‐25 and stained for (A) IL‐25R ( n = 5, three independently differentiated cell lines) or assessed for (B) IL‐25R mRNA levels ( n = 3). (C) Surface expression of IL‐25R on CRTh2 + Th2 cells treated (24 h) with a CRTh2 agonist (DK‐PGD 2 ) or IL‐2 ( n = 4). Statistical significance determined by paired t test within each time point (A, B) or between conditions (C), * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: Cell Culture, Staining, Expressing

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: IL‐25 slows re‐expression of CRTh2 on Th2 cells following TCR activation. (A) Differentiated CRTh2 + Th2 cells cultured with IL‐2, αCD3 and αCD28 exhibited time dependent loss of surface CRTh2 expression ( n = 4, two independently differentiated cell lines). (B–G) CRTh2 + Th2 cells were cultured with IL‐2, αCD3 and αCD28 (24 h) and then placed with IL‐2 in the presence or absence of IL‐25 (another 24 h). % of cells expressing (B) CRTh2, (D) CD62L or (F) CCR4 and mean fluorescent intensity (MFI) for (C) CRTh2, (E) CD62L and (G) CCR4 were quantified ( n = 6, two independently differentiated lines). Statistical significance was determined by paired t test (B–G), * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: Expressing, Activation Assay, Cell Culture

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A) Western blot showing the expression of GPR44 and β-actin in purified 1° WT and Gpr44 −/− AML cells. GPR44 antibody detects glycosylated (63 kDa) and unglycosylated (33 and 50 kDa) and forms. (B) CD45.2- and Ai14 TdTomato -recipient mice were maintained on Se-S diet for 4 weeks before transplantation until the endpoint. 2° transplantation was done retro-orbitally with 1° CD45.1 WT or CD45.2 Gpr44 −/− AML donor cells to CD45.2= and Ai14 TdTomato -recipient mice, respectively; 3 weeks later, mice were euthanized; blood, bone marrow, and spleen were sampled (n = 8 in each group). (C) CBC analysis of Se-S AML mice in (B). (D and E) Counts of AML cells in the Lin − population in the bone marrow (D) and spleen (E) of Se-S AML mice in (B). (F and G) Counts of LICs (WT: CD45.1 + Lin − Sca-1 − c-Kit + , see also ; Gpr44 −/− : RFP − Lin − Sca-1 − c-Kit + , see also ) in the bone marrow (F) and spleen (G) of Se-S AML mice in (B). (H) Survival curve of recipient mice with competitive 2° transplantation of WT (4 × 10 5 ), WT + Gpr44 −/− (2 × 10 5 : 2 × 10 5 ), or Gpr44 −/− (4 × 10 5 ) AML donor cells (n = 6–9 in each group). (I) Progression of WBCs in the peripheral blood of recipient mice secondarily transplanted with WT or Gpr44 −/− AML donor cells (n = 8–9 in each group). (J) Purified 1 WT and Gpr44 −/− AML cells were plated in methylcellulose medium (2,500 cells/well, 4 replicates). CFUs were counted on day 8. (K) Representative image of colony growth from purified 1 WT and Gpr44 −/− AML cells. Scale bar, 100 μm. (L) Comparison of GPR44 expression in blood cancers compared with normal subjects. (M) Comparison of GPR44 expression in AML FAB subtypes including M0, M1, M2, M3, M4, M4Eo, M5, M6, and M7. (L and M) Data were generated from the ONCOMINE database. (L) Each point represents a comparison of the study between the cancer population and normal population. (M) Each point represents a comparison of the study of one FAB subtype with the other subtypes. Plots were generated using the −log10 (p value) and the fold change in expression. p values were obtained by t test of the mean values. An absolute fold change of 1.5 (red line) or higher is considered significant. Data shown are mean ± SEM per group; *p < 0.05, **p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Western Blot, Expressing, Purification, Transplantation Assay, Comparison, Generated

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A) Scheme for sequencing analysis. Bone marrow cells were isolated from mice secondarily transplanted with 1° WT or Gpr44 −/− AML donor cells and LICs were flow cytometrically sorted following RBC lysis, Lin − selection, and Sca-1 and c-Kit staining. LICs (4 × 10 5 ) were used for RNA sequencing. Differential gene expression analysis, IPA, and GSEA were performed to compare WT and Gpr44 −/− LICs (n = 3 in each group). (B) Heatmap for top 100 most differentially regulated genes in WT and Gpr44 −/− LICs. (C) Volcano plot for differential gene expression analysis between WT and Gpr44 −/− LICs (D) Heatmap of indicated pathways in WT and Gpr44 −/− LICs as analyzed by IPA. Z scores were plotted. (E) Expression of Kras assessed by qPCR analysis in WT or Gpr44 −/− AML cells isolated from bone marrow (left) and spleen (right) of 2° AML-recipient mice. Data were normalized to WT AML cells and 18S rRNA expression (n = 4–8 biological replicates). (F) Western blot showing the expression of KRAS in WT or Gpr44 −/− AML cells isolated from 2° AML-recipient mice. (G) Western blot showing the expression of MAPK signaling pathway components including P-C-RAF, RAF-1, P-MEK3/6, MEK1/2, P-ERK, ERK1/2, P-C-JUN,and β-actin in WT or Gpr44 −/− AML cells isolated from 2° AML-recipient mice (n = 5–8 biological replicates in each group). (H) Western blot showing the expression of P53 in WT or Gpr44 −/− AML cells isolated from 2° AML-recipient mice. (F and H) Densitometry was done by normalizing to WT AML cells and relative to β-actin (n = 3–5 biological replicates in each group). Data shown are mean ± SEM per group; *p < 0.05, **p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Sequencing, Isolation, Red Blood Cell Lysis, Selection, Staining, RNA Sequencing, Gene Expression, Expressing, Western Blot

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A) Scheme showing the treatment of BAY293 and CCK8 assay on unpurified Gpr44 −/− AML cells. Frequency of AML cells was above 95%. (B) Comparison of the viability of unpurified Gpr44 −/− AML cells treated with BAY293 (0, 5, 10, and 15 μM, n = 3) for 24 or 48 h. Data were normalized to 0 μM (24 h) treatment. **p < 0.01, comparison between concentrations in the 24 and 48 h time points analyzed by Student’s t test; ## p < 0.01, comparison between time points analyzed by two-way ANOVA followed by appropriate post hoc test (Bonferroni correction). (C) Scheme showing the treatment of BAY293 and live/dead cell measurement on purified Gpr44 −/− AML cells. CD45.2 Gpr44 −/− AML cells were transplanted into CD45.1 recipients; purification of Gpr44 −/− AML cells isolated from spleen was done by CD45.1-positive magnet selection kit. (D and E) Frequency (D) and count (E) of live cells in purified Gpr44 −/− AML cells treated with 15 μM BAY293 for 48 h (n = 3). (F) Western blot showing the expression of P-MEK3/6 and P-ERK in purified 1° Gpr44 −/− AML cells transduced with pLV hU6-sgRNA KRAS hUbC-dCas9-KRAB-T2a-GFP virus. (G) Western blot showing the expression of KRAS in purified 2° Gpr44 −/− AML cells transduced with pLV hU6-sgRNA KRAS hUbC-dCas9-KRAB-T2a-GFP virus. Densitometry was done by normalizing to control group and relative to β-actin (n = 3–4 in each group). (H) Survival curve of AML mice tertiarily transplanted with purified KRAS KD Gpr44 −/− AML cells (n = 7). (I and J) Survival analysis of mice tertiarily transplanted PD98059-treated Gpr44 −/− AML cells. Gpr44 −/− AML cells were cultured ex vivo with or without 100 μM PD98059 for 24 h and then retro-orbitally transplanted into mice (n = 6 per group). Survival was followed up for 60 days post transplantation. (K) Western blot showing the expression of P-ERK and P53 in Gpr44 −/− AML cells treated with PD98059 (0, 10, 50, and 100 μM) for 24, 48, and 72 h. (L and M) Representative image of colony growth from purified 2° Gpr44 −/− AML cells (2,500 cells/well, n = 4 for biological replicate, n = 2 for technical replicate) treated with 100 μM PD98059 and 15 μM BAY293 in methylcellulose medium. Scale bar, 100 μm. (N) Counts of CFUs from purified 2° Gpr44 −/− AML cells treated with 100 μM PD98059 and 15 μM BAY293 in methylcellulose medium. CFUs were counted on day 8. Data shown are mean ± SEM per group; *p < 0.05, ** p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: CCK-8 Assay, Comparison, Purification, Isolation, Selection, Western Blot, Expressing, Transduction, Virus, Control, Cell Culture, Ex Vivo, Transplantation Assay

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet: (A–D) Expression of genes including PI3K ( Pik3ca , Pik3cb , Pik3cd ) (A), AKT ( Akt1 , Akt2 ) (B), PKC ( Prkca , Prkcb , Prkcg ) (C), and PKA ( Prkacg ) (D) assessed by qPCR analysis in WT or Gpr44 −/− AML cells isolated from spleens of 2 AML-recipient mice. Data were normalized to WT AML cells and Gapdh expression (n = 8–10 biological replicates in each group). (E) Western blot showing the expression of Phospho-PI3K, PI3K, Phospho-mTOR, mTOR, Phospho-AKT, AKT, Phospho-P70-S6K, P70-S6K, and β-actin in purified 1° WT and Gpr44 −/− AML cells. (F) Western blot showing the expression of Phospho-PTEN, PTEN, Phospho-4EBP1, 4EBP1, and β-actin in WT or Gpr44 −/− AML cells isolated from spleens of 2° AML-recipient mice (n = 5 biological replicates in each group). (G) Viability of purified 1 Gpr44 −/− AML cells treated with 150 μM ARN509, 10 μM LY294002, 10 nM Wortmannin, 10 nM Sapanisertib, 250 nM Torin1 for 24 h. Data were normalized and compared with vehicle treatment. (H and I) Flow cytometric analysis of Phospho-P70-S6K (H) and Phospho-4E-BP1 (I) in CD45.1 + 1° Gpr44 −/− AML cells treated with 10 μM LY294002, 10 nM Wortmannin, 10 nM Sapanisertib, 250 nM Torin1 for 24 h. MFI was summarized (n = 4). (J) Counts of CFUs of purified 1° Gpr44 −/− AML cells treated with 10 μM LY294002, 10 nM Sapanisertib, and 2.5 μM LY2584702 in methylcellulose medium (2,500 cells/well, n = 3 for technical replicate) CFUs were counted on day 8. (K) Representative image of colony growth in (J). Scale bar, 100 μm. Data shown are mean ± SEM per group; *p < 0.05, ** p < 0.01.

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Expressing, Isolation, Western Blot, Purification

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Prostanoid CRTH2 (human) membrane preparation, in CHO-K1 cells , Perkin Elmer , Cat# ES-561-M400UA.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry