Journal: PLOS Pathogens

Article Title: Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis

doi: 10.1371/journal.ppat.1011165

Figure Lengend Snippet: (A) Mouse peritoneal macrophages were infected with GFP expressing H37Rv (Rv-GFP) at 1:10 MOI followed by treatment with BBR (20 μg/ml). At different time points, cells were analysed by flow cytometry. Graph represents the GFP fluorescence at indicated time points with and without BBR treatment. (B-C) Mouse peritoneal macrophages were infected with M . tb at MOI of 1:10 followed by treatment with BBR (20 μg/ml) for 48 h followed by apoptosis analysis via flow cytometry. (B) Representative dot plots and (C) Percentage of apoptotic cells with and without BBR (20μg/ml) treatment. (D) Immunoblots depicting the phosphorylation of indicated transcription factors (NFkB and STAT3) in uninfected and infected mouse peritoneal macrophages with or without BBR treatment. (E-G) Infected murine peritoneal macrophages were surface stained with antibodies against CD11b (APC/Cy7), CD40 (PE) and CD86 (PerCPCy5.5) followed by flow cytometry. (E) Expression of CD11b on the surface of infected macrophages. Percentage of (F) CD11b + CD40 + and (G) CD11b + CD86 + infected macrophages with and without BBR treatment. (H) Expression of chemokines and cytokines in M . tb infected macrophages at 24h pi with and without BBR (20 μg/ml) treatment. (I) Percentage of CD4 + and CD8 + T cells expressing CD69 in the infected and BBR (10μg/ml) treated splenocytes. (J) Representative overlay plots and (K) percentage of RvGFP infected macrophages co-cultured with M . tb specific and BBR treated splenocytes. (L) PMA- activated THP1 macrophages were infected with H37Rv at 1:10 MOI followed by treatment with INH (1 μg/ml), BBR (20 μg/ml) or both for 48 h pi after which cells were lysed for CFU enumeration. (M) Experiment L was repeated in mouse peritoneal macrophages. (N) Mouse peritoneal macrophages were infected with MDR (Jal 2261) and XDR (MYC431) clinical strains of M . tb followed by treatment with 20 μg/ml of BBR. Cell lysates were plated for CFU enumeration 48 h pi. (O) Schematic representation of the murine model of infection. C57BL/6 mice were infected with low dose of H37Rv (~110 CFU per lung) and after 15 days of disease establishment, mice were treated with either INH (100 mg/L), BBR (4 mg/kg) or both for 45 days followed by CFU enumeration and immune profiling. (P) Histopathology of infected lungs with arrows indicating the granulomatous lesions. (Q) Immunopathology score of the infected lungs. Bacterial burden in the (R) lungs and the (S) spleen of infected animals. (T) Diagrammatic representation of the infection model. Bacterial burden in (U) the lungs and (V) the spleen of mice infected with MDR strain of M . tb . Bacterial load in (W) the lungs and (X) the spleen of mice infected with XDR strain of M . tb . Data is representative of two independent experiments. The data values represent mean ± SD (n = 3–5). *p<0.05, **p<0.005, ***p<0.0005.

Article Snippet: Anti-human/anti-mouse: STAT3, p-STAT3, STAT4, p-STAT4, AKT, p-AKT, FOXO1, p-FOXO1, NFκB, p-NFκB, BLIMP1 and β-Tubulin from Cell Signaling Technology.

Techniques: Infection, Expressing, Flow Cytometry, Fluorescence, Western Blot, Staining, Cell Culture, Histopathology

Journal: PLOS Pathogens

Article Title: Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis

doi: 10.1371/journal.ppat.1011165

Figure Lengend Snippet: Splenocytes isolated from M . tb infected mice were ex vivo stimulated with M . tb CSA and treated with BBR (10 μg/ml) for 48 h. Ex vivo stimulated splenocytes were surface stained with α-CD3 (Pacific Blue), α-CD4 (PerCPCy5.5), α-CD8 (APCCy7), α-CD69 (PE), α-CD103 (PECy7), α-CD62L (APC) and α-CD44 (FITC). (A) Representative dot plots and the percentage of (B) CD4 + T NAIVE cells, (C) CD4 + T EM cells, (D) CD4 + T CM cells and (E) CD4 + T RM cells after BBR treatment. (F-I) To analyse the activation status of key signaling molecules and transcription factors, the cells were stained with α-CD3 (Pacific Blue) and α-CD4 (PerCPCy5.5) followed by intracellular staining with antibodies against p-AKT and p-FOXO1 (see ). (F) Representatives FACS scatter plots and (G) the percentage of CD4 + T cells expressing pAKT. (H&I) Representative scatter plots and the percentage of CD4 + T cells expressing p-FOXO1. (J&K) Ex vivo stimulated splenocytes were treated with BBR (10 μg/ml), AKTi (2.5μM) or both for 48 h followed by surface staining with α-CD3 (Pacific Blue), α-CD4 (PerCPCy5.5), α-CD69 (PE), α-CD103 (PECy7). (J) Percentage of CD4 + T EM and (K) CD4 + T RM cells. (L-S) Stimulation of transcription factors involved in memory responses were examined for which the cells were stained with α-CD3 (Pacific Blue) and α-CD4 (PerCPCy5.5) followed by intracellular staining with antibodies against p-STAT3, p-STAT4, BlIMP-1 and p-NFκB (see ). Representatives FACS scatter plots and percentage of CD4 + T cells expressing (L&M) p-STAT3, (N&O) p-STAT4, (P&Q) Blimp-1 and (R&S) p-NFκB. (T) Expression of cytokines in M . tb specific T cells with or without BBR treatment. (V-X) Ex vivo stimulated splenocytes isolated from M . tb infected mice were treated with BBR (10 μg/ml), 2-Deoxy-D-glucose (2DG; 200 mM), both or left untreated for 24 h. (U) L-Lactate present in the supernatant of treated splenocytes. (V) Percentage of CD4 + IFNγ + T cells and (W) CD4 + IL17 + T cells. The data values represent mean ± SD (n = 3–4). *p<0.05, **p<0.005, ***p<0.0005.

Article Snippet: Anti-human/anti-mouse: STAT3, p-STAT3, STAT4, p-STAT4, AKT, p-AKT, FOXO1, p-FOXO1, NFκB, p-NFκB, BLIMP1 and β-Tubulin from Cell Signaling Technology.

Techniques: Isolation, Infection, Ex Vivo, Staining, Activation Assay, Expressing

Journal: PLOS Pathogens

Article Title: Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis

doi: 10.1371/journal.ppat.1011165

Figure Lengend Snippet: In response to M . tb infection, BBR establishes long-lived, host protective resident memory T cells (TRM) at the site of infection. BBR enhances the effector functions of T lymphocytes by enhancing CD69 expression, directing metabolic flux towards glycolysis, activation of key host protective signaling pathways, and pro-inflammatory immune responses. BBR enriches pathways essential for the establishment and maintenance of memory T cells. BBR upregulates NOTCH3 which directs PTEN to simultaneously inhibit AKT and activate STAT signaling. AKT inhibition further decreases FOXO1 phosphorylation thereby enhancing its nuclear retention. BBR-mediated enhancement of activated STAT4 and STAT3-mediated BLIMP1 signaling axis further results in heightened expression of TRM-specific genes for long-term protection against M.tb infections.

Article Snippet: Anti-human/anti-mouse: STAT3, p-STAT3, STAT4, p-STAT4, AKT, p-AKT, FOXO1, p-FOXO1, NFκB, p-NFκB, BLIMP1 and β-Tubulin from Cell Signaling Technology.

Techniques: Infection, Expressing, Activation Assay, Inhibition

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: ANKRD22 was highly expressed in CD11b + Ly6G + Ly6 C low cells. (A) IHC staining of ovarian cancer and normal ovarian tissues from our center showed that no ANKRD22 expression was observed in normal ovarian cells or ovarian tumor cells, while ANKRD22 was expressed in the normal stroma and cancerous ovarian stromal tissues. Scale bars are 200 µm and 50 µm. (B). The BioGPS database showed the expression level of ANKRD22 in different immune cells (n=262) and human bone marrow cells at different stages (n=745); myeloid cells and their precursors, neutrophils, monocytes may express ANKRD22 at a high level. (C). FACS combined with RT-qPCR showed that Ankrd22 expression was the highest in mouse BM-derived CD11b + Ly6G + Ly6C low cells than monocytes and macrophages, while Ankrd22 was almost undetectable in B cells and plasma cells. (D). The proportion of infiltrating immune cells in human ovarian cancer tissue increased to different degrees. The above data were provided by the lymphocyte infiltration module of the TISIDB. (E). The expression level of Ankrd22 in CD11b + Ly6G + Ly6C low cells from the bone marrow, spleen or peripheral blood of the wild-type C57BL/6 mice was detected by RT-qPCR. n=3, *p<0.05, ***p<0.001. Two-sample Student’s t-test. (F). BM-derived CD11b + Ly6G + Ly6C low cells and BM-derived CD11b + Ly6G - Ly6C high cells were sorted by FACS, and the expression level of Ankrd22 in these cells was detected by RT-qPCR, respectively. n=3, ***p<0.001. Two-sample Student’s t-test. BM, bone marrow; BMC, bone marrow cells; FACS, fluorescenceactivated cell sorting; IHC, immunohistochemical; TISIDB, Tumor-Immune System Interaction Database.

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Derivative Assay, FACS, Immunohistochemical staining

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: The tumor microenvironment affected the expression of Ankrd22 in BM-derived CD11b + Ly6G + Ly6 C low cells. (A) RT-qPCR was used to detect the expression level of Ankrd22 in CD11b + Ly6G + Ly6C low cells cultured under hypoxia, glucose deprivation, lactic acid accumulation, TGF-β, IFN-γ, ROS and CPT-11 inducing senescence，n=3, **p<0.01, ***p<0.001. (B) A subcutaneous ovarian cancer model was established in C57BL/6 mice. BM-derived CD11b + Ly6G + Ly6C low cells and subcutaneous tumor tissues derived CD11b + Ly6G + Ly6C low cells were sorted by FACS from tumor-bearing mice after 8 weeks, and the Ankrd22 expression level in CD11b + Ly6G + Ly6C low cells was detected by RT-qPCR. CD11b + Ly6G + Ly6C low cells from the bone marrow of wild-type mice were used as a control. *p<0.05, ***p<0.001. Two-sample Student’s t-test. (C) Ovarian tissues were collected from patients with and without ovarian cancer undergoing ovarian tissue excision. CD11b + HLA-DR - CD14 - CD15 + cells were sorted by FACS after digestion, and the expression level of ANKRD22 were detected by RT-qPCR (n=6 for non-ovarian cancer and n=20 for ovarian cancer). ***p<0.001, two-sample Student’s t-test. BM, bone marrow; FACS, fluorescenceactivated cell sorting.

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Cell Culture, FACS

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: Knockout of Ankrd22 enhanced the immunosuppressive activity of PMN-MDSCs. (A) Diagram of the construction of the Ankrd22 -knockout C57BL/6 mouse model. (B). Bone marrow was collected from KO mice and WT mice to prepare single-cell suspensions, and then CD11b + Ly6G + Ly6C low cells were sorted by FACS. PCR and agarose gel electrophoresis were used to detect Ankrd22 . M: marker. n=3. Ankrd22 in gastric epithelial cells was used as a positive control. (C). The HPA database showed the expression level of ANKRD22 among human blood and immune cells based on flow sorted. Human peripheral blood neutrophils and mononuclear cells were extracted following the instruction of human peripheral blood neutrophil/mononuclear cell isolation kit, and the expression of ANKRD22 was measured by RT-qPCR and western blotting. The lysate of the ANKRD22-overexpressing (OE) 293 T cells as a positive control. n=3, **p<0.01. Two-sample Student’s t-test. (D, E) FCM was used to detect the percentage of proliferating CFSE-labeled T cells after coculture with CD11b + Ly6G + Ly6C low cells which were treated with 100 ng/mL GM-CSF and 100 ng/mL IL-6 for 96 hours. CFSE fluorescence intensity of T cells were showed. n=3, **p<0.01. Two-sample Student’s t-test. (F). The content of IL-2 in the supernatant of cocultured cells was detected by ELISA. n=3, **p<0.01. Two-sample Student’s t-test. CFSE, carboxyfluorescein diacetate succinimidyl ester; FACS, fluorescenceactivated cell sorting; FCM, flow cytometry; HPA, Human Protein Atlas; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; WT, wild type.

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Knock-Out, Activity Assay, Agarose Gel Electrophoresis, Marker, Positive Control, Expressing, Cell Isolation, Quantitative RT-PCR, Western Blot, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, FACS, Flow Cytometry, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: Knockout (KO) of Ankrd22 promoted the proliferation of ovarian cancer cells by enhancing the immunosuppressive ability of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in transplanted tumor mouse models. (A) 5×10 6 ID8 cells/mouse mixed with 10×10 6 BM-derived CD11b + Ly6G + Ly6C low cells from Ankrd22 +/+ or Ankrd22 -/- C57BL/6 mice, were suspended in Matrigel and injected into the axilla of WT C57BL/6 mice. Tumor diameters were measured with a Vernier caliper once a week for 8 weeks. The growth of subcutaneous tumors in mice was recorded. At the end of the experiment, the subcutaneous tumor weight was determined. n=4, **p<0.01, ***p<0.001. Two-sample Student’s t-test. (B). 5×10 6 ID8 cells/ mouse were mixed with 10×10 6 neutrophils from mouse peripheral blood and injected into the axilla of WT C57BL/6 mice. Tumor diameters were measured with a Vernier caliper once a week for 8 weeks. The growth of subcutaneous tumors in mice was recorded. At the end of the experiment, the subcutaneous tumor weight was determined. n=4, NS: p>0.05. Two-sample Student’s t-test. (C). Subcutaneous tumor tissues were collected from KO and WT mice, and intracellular Arg-1, iNOS and IDO on CD11b + Ly6G + Ly6C low cells were detected by flow cytometry (FCM) after stimulation with 81.0 nM PMA and 1.34 µM ionomycin. n=4, *p<0.05, **p<0.01, ***p<0.001. (D) FCM was used to detect PD-L1 on CD11b + Ly6G + Ly6C low cells derived from the bone marrow, spleen, peripheral blood or subcutaneous tumor tissues of the tumor-bearing mice in WT and KO groups. ***p<0.001. Two-sample Student’s t-test. (E.) FCM was used to detect the percentage of proliferating carboxyfluorescein diacetate succinimidyl este (CFSE)-labeled T cells after coculture with CD11b + Ly6G + Ly6C low cells from mice tumor tissues without treatment of 100 ng/mL GM-CSF and 100 ng/mL IL-6 for 96 hours. CFSE fluorescence intensity of T cells were showed. n=3, **p<0.01. Two-sample Student’s t-test. (F). The content of IL-2 in the supernatant of cocultured cells was detected by ELISA. n=3, **p<0.01. Two-sample Student’s t-test. (G–I). CD11b + Ly6G + Ly6C low cells from the tumor-bearing mice were cultured with DMEM containing 2-NBDG under 100 ng/mL GM-CSF and 100 ng/mL IL-6 stimulation for 7 days. The intensity of green fluorescence in the PMN-MDSCs was detected by immunofluorescence microscopy. The FCM results show the percentage of PMN-MDSCs with FITC fluorescence. n=3, ***p<0.001. BM, bone marrow; FITC, fluorescein isothiocyanate.

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Knock-Out, Derivative Assay, Injection, Flow Cytometry, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunofluorescence, Microscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: The immunosuppressive activity of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) Ankrd22-/- was enhanced by Wdfy1. (A) RNA sequencing. Bone marrow (BM)-derived CD11b + Ly6G + Ly6C low cells from Ankrd22 -/- and Ankrd22 +/+ mice which sorted by fluorescenceactivated cell sorting (FACS) were checked. The heatmap shows mainly changed genes between these two groups, n=3. (B). BM-derived CD11b + Ly6G + Ly6C low cells from Ankrd22 -/- and Ankrd22 +/+ mice were cultured with 100 ng/mL GM-CSF and 100 ng/mL IL-6, and the cell supernatants were collected for cytokine chip analysis after normoxic or hypoxic culture for 24 hours. The heatmap shows changes in the expression of cytokines in the supernatants of PMN-MDSCs Ankrd22-/- and PMN-MDSCs Ankrd22+/+ . n=3. (C). Heatmap analysis. RNA sequencing showed that the expression of the transcription factors Wdfy1 and Mlxip was significantly upregulated in BM-derived CD11b + Ly6G + Ly6C low cells from Ankrd22 -/- mice. (D). PMN-MDSCs from wild-type (WT) mice were transfected with Wdfy1 and maintained with 100 ng/mL GM-CSF and 100 ng/mL IL-6, and then immunosuppressive molecules, such as Arg1, Ido, Inos and Pdl1, in the PMN-MDSCs were detected by RT-qPCR. n=3, *p<0.05，***p < 0.001. Two-sample Student’s t-test. (E). BM-derived CD11b + Ly6G + Ly6C low cells from WT mice cultured with 100 ng/mL GM-CSF and 100 ng/mL IL-6 and 2-NBDG was infected with lentivirus overexpressing Mlxip . The ratio of 2-NBDG in the PMN-MDSCs was detected by flow cytometry (FCM) and the graph showed the exogenous glucose uptake ability of PMN-MDSCs. n=3, **p < 0.01. Two-sample Student’s t-test. OE, overexpression.

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Activity Assay, Derivative Assay, RNA Sequencing Assay, FACS, Cell Culture, Expressing, Transfection, Quantitative RT-PCR, Infection, Flow Cytometry, Over Expression

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: An ANKRD22 agonist reversed the immunosuppressive activity of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). (A) Schematic diagram of ANKRD22-activating small-molecule compounds. (B) Human THP-1 cells were cocultured with agonist at concentrations of 0, 0.1, 0.5, 1, 5 and 10 µM for 24 hours. The mean fluorescence intensity of Ca 2+ was measured to evaluate whether the small-molecule compound was effective. (C) Colorectal cancer LS-174T cells were cocultured with agonists at concentrations of 0, 0.1, 0.5, 1, 5 and 10 µM for 24 hours. The expression level of ANKRD22 was detected by western blotting. (D) BM-derived CD11b + Ly6G + Ly6C low cells from wild-type (WT) mice, stimulated with 100 ng/mL GM-CSF and 100 ng/mL IL-6 (PMN-MDSCs Ankrd22+/+ ), were cocultured with actived carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled spleen-derived T cells in the presence of 1.0 µM ANKRD22-activating small-molecule compound for 96 hours. DMSO was added to the FCM culture medium as a control. Flow cytometry (FCM) showed the effect of PMN-MDSCs on T-cell proliferation after addition of the activating small-molecule compound. ELISA was used to detect IL-2 concentration (pg/mL) in the supernatant of PMN-MDSCs cocultured with T cells. n=3, ***p<0.001. Two-sample Student’s t-test. (E) PMN-MDSCs Ankrd22+/+ were infected with recombinant lentivirus overexpressing Ankrd22 . FCM shows the effect of PMN-MDSCs on T-cell proliferation after infection. ELISA was used to detect the IL-2 concentration in the cell supernatant of PMN-MDSCs cocultured with T cells. n=3, **p<0.01, ***p<0.001. Two-sample Student’s t-test. (F) PMN-MDSCs were treated with an ANKRD22 agonist for 72 hours in vitro, and the results showed that the levels of Arg-1, iNOS, IDO and PD-L1 were reduced in PMN-MDSCs which were with ANKRD22 agonist. n=3, *p<0.05, **p<0.01, ***p<0.001. Two-sample Student’s t-test. BM, bone marrow.

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Activity Assay, Derivative Assay, Fluorescence, Expressing, Western Blot, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Infection, Recombinant, In Vitro

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: ANKRD22 knockout increases CCR2 expression and affects the clinical characteristics and prognosis of ovarian cancer. (A, B) BM-derived CD11b + Ly6G + Ly6C low cells from Ankrd22 -/- and Ankrd22 +/+ mice were mixed with ID8 cells to construct the tumor-bearing mouse model on WT mice. After tumor formation in mice, the BM, spleen, peripheral blood and subcutaneous tumor tissues were collected to isolate CD11b + Ly6G + Ly6C low cells by fluorescenceactivated cell sorting (FACS), and the expression of CCR2 was determined by flow cytometry (FCM). n=3, ***p<0.001. Two-sample Student’s t-test. (C) According to the expression level of ANKRD22 , patients with ovarian cancer were divided into a high-expression group and a low-expression group, and the numbers of neutrophils, monocytes, platelets and lymphocytes in the peripheral blood were determined. (A) Neutrophil-to-lymphocyte ratio (NLR), n=20, **p<0.01; (B) Monocyte-to-lymphocyte ratio (MLR), n=20, *p<0.05; (C) Platelet-to-lymphocyte ratio (PLR), n=20, *p<0.05. Two-sample Student’s t-test. BM, bone marrow; WT, wild type.

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Knock-Out, Expressing, Derivative Assay, Construct, FACS, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet: The correlation between different ANKRD22 expression levels andclinicopathological features of patients with ovarian cancer

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques: Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: ANKRD22 is a potential novel target for reversing the immunosuppressive effects of PMN-MDSCs in ovarian cancer

doi: 10.1136/jitc-2022-005527

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were as follows: rabbit anti-human β-actin (CST), rabbit anti-human β-tubulin (CST), and house-made mouse anti-human ANKRD22 (clone number: 1A8).

Techniques:

Journal: Genome Medicine

Article Title: Tumor microenvironment remodeling after neoadjuvant immunotherapy in non-small cell lung cancer revealed by single-cell RNA sequencing

doi: 10.1186/s13073-023-01164-9

Figure Lengend Snippet: scRNA-seq analysis of NSCLC during therapy. A Scheme of the overall study design. B Uniform manifold approximation and projection (UMAP) plot of all cells colored by major cell types according to canonical markers. C Bar plots indicating the proportion of major cell lineages in each patient. D Boxplot showing cellular fractions of T, natural killer (NK), B, myeloid cells and neutrophils in TN ( n = 3), MPR ( n = 4), and NMPR ( n = 8) patients. Center line indicates the median value, lower and upper hinges represent the 25th and 75th percentiles, respectively, and whiskers denote 1.5× interquartile range. Each dot corresponds to one sample. All adjusted P values were larger than 0.05. One-sided unpaired Wilcoxon test was used, and the P values were adjusted by the FDR method. E Representative images of immunohistochemistry (IHC) staining of canonical surface markers for T (CD3), NK (CD56), and B (CD20) cells in a TN (S01b), MPR (P06), and NMPR (P07) patient, respectively. F Quantification of fractions of T, NK, and B cells from the IHC images. One-sided unpaired Wilcoxon test was used

Article Snippet: We performed the IHC by using CD3 rabbit anti-human antibody (Biolynx, BX50022), CD20 mouse anti-human antibody (Dako, M0755), and CD56 mouse anti-human antibody (Cell Signaling Technology, 3576S).

Techniques: Immunohistochemistry

Journal: PLOS ONE

Article Title: Novel Bruton’s tyrosine kinase inhibitor TAS5315 suppresses the progression of inflammation and joint destruction in rodent collagen-induced arthritis

doi: 10.1371/journal.pone.0282117

Figure Lengend Snippet: (a) Phosphorylation of BTK induced by RANKL stimulation (100 ng/mL) for 24 h in M-CSF-induced murine macrophages. Macrophages were simultaneously subjected to TAS5315 and RANKL stimulation. (b) Nuclear translocation of NFATc1 induced by RANKL stimulation (100 ng/mL) for 24 h in M-CSF-induced murine macrophages. M-CSF-induced murine macrophages were simultaneously subjected to TAS5315 and RANKL stimulation. The figures of immunoblot experiments (a, b) represent the data of three independent experiments. (c, d) Human osteoclast differentiation was induced by RANKL (66 ng/mL) and M-CSF (33 ng/mL) in the presence of DMSO, TAS5315, or anti-RANKL antibody (0.1 μg/mL) for 4 days. (c) Representative images of TRAP-positive osteoclasts. (d) TRAP-positive cells with 3 or more nuclei (cell size ≥50 μm) were counted as osteoclasts. (e) CTX-I levels in the supernatant of human osteoclasts treated with M-CSF (33 ng/mL) and RANKL (66 ng/mL) in the presence of DMSO, TAS5315, or anti-RANKL antibody (0.1 μg/mL) for 14 days. (f, g) Pit formation by mouse osteoclasts treated with RANKL (25 ng/mL) and M-CSF (50 ng/mL) in the presence of DMSO, TAS5315, or anti-RANKL antibody (0.1 μg/mL) for 16 days. (f) Representative images of pit formation by mouse osteoclast (DMSO, TAS5315 100 nM, anti-RANKL antibody). (g) Quantitative analysis of the relative pit formation area by mouse osteoclasts. Experiments (c–g) were performed in triplicates and the numerical data (d, f, g) are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001 compared with DMSO group (Dunnett test for TAS5315 groups or Student’s t-test for anti-RANKL antibody).

Article Snippet: The following antibodies were used: rabbit anti-human phosphorylated BTK (tyrosine 223) monoclonal antibody (EP420Y) (ab68217), mouse anti-human p84 monoclonal antibody (ab487) (Abcam, Cambridge, UK), mouse anti-human BTK monoclonal antibody (611117), fluorescein isothiocyanate-conjugated rat anti-mouse CD45R/B220 monoclonal antibody (553088), phycoerythrin-cyanine 7-conjugated anti-mouse CD45R/B220 monoclonal antibody (552772), allophycocyanin-conjugated rat anti-mouse CD86 monoclonal antibody (558703), brilliant violet 421-conjugated rat anti-mouse I-A/I-E monoclonal antibody (562564) (BD Biosciences, San Jose, CA, USA), APC-conjugated Armenian hamster anti-mouse CD69 monoclonal antibody (17-0691-82; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-human G3PDH/GAPDH polyclonal antibody (2275-PC-100; Trevigen, Gaithersburg, MD, USA), rabbit anti-mouse BTK monoclonal antibody (8547), rabbit anti-human phosphorylated PLCγ2 (tyrosine 1217) polyclonal antibody (3871), rabbit anti-human PLCγ2 polyclonal antibody (3872), rabbit anti-human phosphorylated AKT (serine 473) monoclonal antibody (4075), rabbit anti-human AKT polyclonal antibody (9272), rabbit anti-human phosphorylated ERK (threonine 185 and tyrosine 187) monoclonal antibody (4370), rabbit anti-human ERK polyclonal antibody (9102) (Cell Signaling Technology, Danvers, MA, USA), mouse anti-human NFATc1 monoclonal antibody (MABS409), and mouse anti-bovine β-tubulin monoclonal antibody (05–661) (Merck Millipore, Darmstadt, Germany).

Techniques: Translocation Assay, Western Blot

Journal: Cancers

Article Title: Genome-Wide Analysis of lncRNA-mRNA Co-Expression Networks in CD133+/CD44+ Stem-like PDAC Cells

doi: 10.3390/cancers15041053

Figure Lengend Snippet: CD133+/CD44+ PDAC cells are highly oncogenic in vivo. 8×105 CD133+/CD44+, CD133−/CD44− or total (unsorted) cells derived from ( A ) MIA PaCa-2 and ( B ) PANC-1 cell lines were inoculated heterotopically into the flanks of NGS mice, and the tumor growth was monitored. CD133+/CD44+ cells show similar growth curves with total cells, whilst the sorted cells fail to grow. Tumor volumes were plotted as mean +/− SEM for each data point retrieved by two independent experiments (N = 8–10 mice/per group/experiment). ( C ) Representative immunohistochemical staining for Ki67 expression in tumors formed by MIA PaCa-2- and PANC-1-derived CD133+/CD44+ and total cells. Scale Bar = 200 μm. ( D ) The corresponding percentages of Ki67-positive cells indicate no statistically significant differences in proliferation rate between CD133+/CD44+ and total cells from both cell lines. Data were collected through three independent experiments.

Article Snippet: Cell proliferation was calculated by IHC, using a mouse anti-human mAb against Ki67 (Cell Signaling Technology, Inc., Danvers, MA, USA) (1:1000 dilution), followed by incubation with an HRP-conjugated anti-mouse secondary Ab (Sigma-Aldrich).

Techniques: In Vivo, Derivative Assay, Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Hyaluronan promotes intracellular ROS production and apoptosis in TNFα-stimulated neutrophils

doi: 10.3389/fimmu.2023.1032469

Figure Lengend Snippet: Total and phospho p38 MAPK in TNFα- and HA-stimulated neutrophils. (A) Western blot showing the phosphorylated and total p38 MAPK protein over time in neutrophils primed with HA (200 μg/mL). Data is representative of 3 independent experiments. (B) Graph shows quantification (mean ± SD) of phospho-p38 normalized to total p38 protein (y-axis) over time (x-axis), n = 3. (C) Western blot showing the phosphorylated and total p38 MAPK protein in neutrophils upon fMLF activation (100 nM, 0 – 2 min) at t = 16 min of priming in the presence (HA + fMLF) or absence (fMLF) of HA (200 μg/mL). Data is representative of 3 independent experiments. (D) Graph shows quantification (mean ± SD) of phospho-p38 normalized to total p38 protein (y-axis) over time (x-axis) during activation with fMLF at t = 16 min of priming with (HA + fMLF) and without (fMLF) HA, n = 3. (E) Abundance of phosphorylated and total p38 MAPK protein over time in neutrophils primed with TNFα (10 ng/mL) and HA + TNFα (200 μg/mL HA). Data is representative of 3 independent experiments. (F) Graph shows quantification (mean ± SD) of phospho-p38 normalized to total p38 (y-axis) over time (x-axis) in TNFα-primed and HA+TNFα co-primed neutrophils, n = 3. Statistical analysis for individual time points was performed using unpaired two-tailed t-test, (B, F) ; *p < 0.05.

Article Snippet: The primary antibodies used were the following: rabbit anti-human p38 mAb (1:2,000; Cell Signalling Technology; #8690), mouse anti-human phospho-p38 mAb (Thr180/Tyr182; 1:2,000; Cell Signalling Technology; #9216).

Techniques: Western Blot, Activation Assay, Two Tailed Test