Journal: Cancer Biomarkers

Article Title: Glutaminase 1 plays critical roles in myelodysplastic syndrome and acute myeloid leukemia cells

doi: 10.3233/CBM-230454

Figure Lengend Snippet: Activity of GLS1 inhibitors in MDS and AML cells. (A, B) MDS and AML cell lines were cultured with the indicated concentrations of CB839 or IPN60090 for 72 h under hypoxia. Cell growth was evaluated using the CellTiter-Glo ™ Luminescent Cell Viability Assay Kit or Cell Counting Kit-8. (C, D, E, F) MDS and AML cell lines were cultured with the indicated concentrations of CB838 or IPN60090 for 24 h. The total amount of NADPH or NADP + was analyzed by using NADP/NADPH Assay Kit-WST. (G) SKM-1 cells were incubated with the indicated concentration of CB839 or IPN60090 for 72 h. Intracellular ATP levels were determined using the “Cell” ATP Assay Reagent Ver. 2 Kit. *** p< 0.001, **** p< 0.0001 were compared to control. ns, not significant. (H) SKM-1 cells were incubated with the indicated concentration of CB839 for 24 h. The extracellular OCR and the extracellular acidification were analyzed.

Article Snippet: The extracellular rate of oxygen consumption (OCR) plate assay kit and glycolytic assay (extracellular acidification) kit were obtained from Dojindo Laboratory and Abcam (Cambridge, UK) and analyzed according to the manufacturer’s instructions.

Techniques: Activity Assay, Cell Culture, Cell Viability Assay, Cell Counting, Incubation, Concentration Assay, ATP Assay, Control

Journal: iScience

Article Title: Citrate shuttling in astrocytes is required for processing cocaine-induced neuron-derived excess peroxidated fatty acids

doi: 10.1016/j.isci.2022.105407

Figure Lengend Snippet: Conditioned media from Tat- and/or cocaine-treated neurons alters astrocytic cholesterol homeostasis SLC25A1 was knocked down (KD) in rat astrocytes, and nontargeting scrambled siRNA (Scr siRNA) was used as a control. Astrocytes were then cultured with cell-free rat neuronal media (NCM) collected from either untreated or control or neurons exposed to Tat and/or cocaine. (A–D) Protein levels were assessed by western analyses for SLC25A1. (A) Representative western blot and (B) and quantification of the protein abundance SLC25A1 KD astrocytes. (C and D) Astrocytes with SCL25A1 knocked down and cultured in the presence of BODIPY 493/503 (green) to label lipids show increased lipid accumulation compared with Scr siRNA upon exposure to Tat/cocaine NCM. (E and F) Scr siRNA and SLC25A1 KD astrocytes treated with control or Tat/cocaine NCM were stained with Filipin III (blue) to visualize the intracellular cholesterol levels. Representative fluorescence microscopy images (E) and quantification of Filipin fluorescence (F) show decreased accumulation of cholesterol in KD SCL25A1 astrocytes exposed to Tat and/or cocaine NCM. (G and H) Extracellular cholesterol and ApoE protein levels were quantified in astrocyte media using ApoE ELISA and Amplex Red cholesterol assay, respectively. Astrocytes exposed to Tat and/or cocaine NCM show increased levels of cholesterol and ApoE, but knocking down SLC25A1 reduced cholesterol and ApoE levels. Data indicate Mean ± SEM; ∗∗∗p < 0.001, ∗p < 0.05; 3 independent coverslips with 15–20 cells/coverslip analyzed.

Article Snippet: Lactate (MAK064, Sigma-Aldrich), LDH (MAK066, Sigma-Aldrich), extracellular cholesterol (Amplex red cholesterol assay kit A12216, Invitrogen), intracellular cholesterol (cholesterol assay kit, cell-based, ab133116 Abcam), and ApoE (Apolipoprotein E human ELISA kit, EHAPOE, Invitrogen) concentrations or activity in media or in cells were measured following the manufacturer’s instructions.

Techniques: Control, Cell Culture, Western Blot, Quantitative Proteomics, Staining, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Amplex Red Cholesterol Assay

Journal: iScience

Article Title: Citrate shuttling in astrocytes is required for processing cocaine-induced neuron-derived excess peroxidated fatty acids

doi: 10.1016/j.isci.2022.105407

Figure Lengend Snippet:

Article Snippet: Lactate (MAK064, Sigma-Aldrich), LDH (MAK066, Sigma-Aldrich), extracellular cholesterol (Amplex red cholesterol assay kit A12216, Invitrogen), intracellular cholesterol (cholesterol assay kit, cell-based, ab133116 Abcam), and ApoE (Apolipoprotein E human ELISA kit, EHAPOE, Invitrogen) concentrations or activity in media or in cells were measured following the manufacturer’s instructions.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Luminescence Assay, Software

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Colocalization and expression of Piezo1 and Piezo2 in EC cells of FC Mice. (A) Immunofluorescence staining of Piezo1, Piezo2 and EC cells in colonic sections from each group. DAPI (blue) indicates nuclei, Piezo2 (green), Piezo1 (purple) and EC cell (red). Scale bar, 50 μ m (magnification, ×200). Yellow arrows indicate co-localization of Piezo1 and ChgA; orange arrows indicate co-localization of Piezo2 and ChgA; white arrows indicate triple co-localization of Piezo1, Piezo2 and ChgA. Percentage of (B) Piezo1 + ChgA + , (C) Piezo2 + ChgA + and (D) Piezo1 + Piezo2 + ChgA + cells as a percentage of total ChgA + cells in control and FC groups; n=5 mice per group. (E) Quantification of Piezo1 and (F) Piezo2 mRNA levels relative to β-actin in the control and FC group; n=8 mice per group. (G) Western blot analysis of Piezo1 and Piezo2 protein expression in the control and FC groups; n=3 mice per group Target proteins and internal controls were detected on separate membranes processed in parallel. The vertical line indicates membrane separation. (H) Co-immunoprecipitation of Piezo1 and Piezo2 in colonic tissues from control and FC model mice, with Piezo1 immunoprecipitated and Piezo2 detected by western blotting. Data are presented as mean ± SD and were analyzed using an unpaired two-tailed Student's t-test. Statistical significance indicated as * P<0.05, ** P<0.01. EC, enterochromaffin cell; FC, functional constipation.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, Membrane, Immunoprecipitation, Two Tailed Test, Functional Assay

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Piezo1/Piezo2 knockdown attenuates expression of Piezos in EC cells of FC mice. (A) Schematic of the experimental timeline for AAV-mediated knockdown. RT-qPCR analysis of (B) Piezo1 and (C) Piezo2 mRNA levels in the control AAV, AAV Piezo1-KD, AAV Piezo2-KD and AAV Piezo1/2-KD groups; n=5-7 mice per group. Data analyzed by one-way ANOVA with Tukey's post hoc test. (D) Immunofluorescence staining of Piezo1, Piezo2 and EC cells in colonic sections from each group. DAPI (blue) indicates nuclei, Piezo2 (green), Piezo1 (purple) and EC cell (red). Scale bar, 50 μ m (magnification, ×200). Yellow arrows indicate co-localization of Piezo1 and ChgA; orange arrows indicate co-localization of Piezo2 and ChgA; white arrows indicate triple co-localization of Piezo1, Piezo2 and ChgA. Percentage of (E) Piezo1 + ChgA + , (F) Piezo2 + ChgA + and (G) Piezo1 + Piezo2 + ChgA + cells as a percentage of total ChgA + cells in control and knockdown groups; n=5 mice per group. Data represent mean ± SD and were analyzed using Kruskal-Wallis test. Statistical significance is indicated as * P<0.05, ** P<0.01, *** P<0.001. ns, not significant; EC, enterochromaffin cell; FC, functional constipation; AAV, adeno-associated virus; KD, knockdown.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Control, Immunofluorescence, Staining, Functional Assay, Virus

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Effects of Piezo1 and Piezo2 knockdown on gastrointestinal motility and colonic sensitivity in FC Mice. (A-H) General effects of the FC model compared with control mice. Data were analyzed using an unpaired two-tailed Student's t-test. (I-P) Effects of individual or simultaneous Piezo1 and Piezo2 knockdown on gastrointestinal motility and sensitivity in FC mice. Data were analyzed by one-way ANOVA with Tukey's post hoc test or Kruskal-Wallis test. (A and I) TGITT, (B and J) Gastric emptying rate, (C, K, E and L) Small intestinal transit rate, (D and M) Gas volume required to reach an AWR score of 3 and AWR scores at gas volumes of 0.25, 0.35, 0.50 and 0.65 ml. (F and N) Histological analysis of colonic tissue in each group; n=3 mice per group. Red arrows highlight regions of glandular necrosis with structural disorganization and green arrows indicate nuclear pyknosis and cytoplasmic dissolution. Scale bar, 100 μ m (left, magnification, ×100) and 10 μ m (right, magnification, ×400). (G and O) Serum levels of VIP in each group, (H and P) Serum levels of SP in each group. Data were analyzed using an unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's post hoc test. n=6-9 mice per group. Data represent mean ± SD. Statistical significance is indicated as * P<0.05, ** P<0.01, *** P<0.001. FC, functional constipation; TGITT, total gastrointestinal transit time; AWR, abdominal withdrawal reflex VIP, vasoactive intestinal peptide; SP, substance P.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Knockdown, Control, Two Tailed Test, Dissolution, Functional Assay

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Effects of Piezo1 and Piezo2 knockdown on 5-HT synthesis, release and SERT expression in FC Mice. (A) Representative immunohistochemical staining of 5-HT 3 and SERT in colonic sections from control and FC mice. Scale bar, 40 μ m (magnification, ×400). Quantification of (B) serum 5-HT and (C) colonic TPH-1 levels in control and FC mice. Data were analyzed using an unpaired two-tailed Student's t-test; n=4-6 mice per group. (D) Immunohistochemical staining of 5-HT 3 and SERT in colonic sections from control AAV, AAV Piezo1-KD, AAV Piezo2-KD and AAV Piezo1/2-KD groups. n=3 mice per group. Scale bar, 40 μ m (magnification, ×400). (E and F) Serum and colonic 5-HT levels in each group, comparing knockdown groups to the control AAV group. (G and H) Relative mRNA levels of SERT and colonic TPH-1 levels in each group, comparing knockdown groups to the control AAV. Data were analyzed by one-way ANOVA with Tukey's post hoc test. n=4-6 mice per group. Data are presented as mean ± SD, with statistical significance indicated as * P<0.05, ** P<0.01, *** P<0.001. 5-HT, 5-hydroxytryptamine; SERT, serotonin transporter; FC, functional constipation; TPH-1, tryptophan hydroxylase-1; AAV, adeno-associated virus; KD, knockdown.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Knockdown, Expressing, Immunohistochemical staining, Staining, Control, Two Tailed Test, Functional Assay, Virus

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Expression changes of Piezo1 and Piezo2 in EC cells following individual and combined knockdown. Representative Western blot analysis of (A) Piezo1 and (B) Piezo2 protein expression in the NC, VC, LV-Piezo1 KD (Piezo1 knockdown), LV-Piezo2 KD (Piezo2 knockdown) and LV-Piezo1/2 KD (combined Piezo1 and Piezo2 knockdown) groups. GAPDH served as the loading control. Target proteins and internal controls were detected on separate membranes processed in parallel. The vertical line indicates membrane separation. Quantification of (C) Piezo1 and (D) Piezo2 mRNA levels relative to GAPDH; n=3 cells per group. Data are presented as mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. Statistical significance indicated as * P<0.05, ** P<0.01, *** P<0.001. EC, enterochromaffin cell; NC, negative control; VC, vector control; KD, knockdown.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Expressing, Knockdown, Western Blot, Control, Membrane, Negative Control, Plasmid Preparation

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Effect of Piezo1 and Piezo2 knockdown on intracellular Ca 2+ levels in ECs. (A) Representative immunofluorescence images of intracellular Ca 2+ levels in different groups. DAPI (blue) was used to stain nuclei and Ca 2+ indicator (green) shows intracellular calcium levels. Merged images display co-localization of nuclei and Ca 2+ fluorescence. Scale bar, 50 μ m (magnification, ×400). (B) Quantification of Ca 2+ fluorescence intensity in each group. (C) Quantification of Ca 2+ fluorescence counts in each group; n=3 cells per group. Data are presented as mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. Statistical significance indicated as * P<0.05, ** P<0.01, *** P<0.001. ns, not significant; EC, enterochromaffin cell.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Knockdown, Immunofluorescence, Staining, Fluorescence

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Effect of Piezo1 and Piezo2 knockdown on 5-HT and TPH-1 levels in enterochromaffin cells. (A) Quantification of 5-HT levels (ng/l) in the different groups. (B) Quantification of TPH-1 enzyme activity (U/l) in each group. n=3 cells per group. Data are presented as mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. Statistical significance indicated as * P<0.05, ** P<0.01, *** P<0.001. ns, not significant; 5-HT, 5-hydroxytryptamine; NC, negative control; VC, vector control; KD, knockdown; TPH-1, tryptophan hydroxylase-1.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Knockdown, Activity Assay, Negative Control, Plasmid Preparation, Control

Journal: International Journal of Molecular Medicine

Article Title: Piezo knockdown reduces 5-hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

doi: 10.3892/ijmm.2025.5619

Figure Lengend Snippet: Mechanism diagram of the study. In functional constipation, Piezo1 and Piezo2 expression is downregulated, reducing Ca 2+ influx and impairing 5-HT synthesis. Consequently, diminished 5-HT release reduces activation of 5-HT 3 receptors, SP levels decrease and VIP levels increase, leading to smooth muscle relaxation. Additionally, heightened SERT activity, exacerbating the deficiency in 5-HT signaling. These disruptions collectively induce pathological alterations in intestinal structure and impaired motility, leading to functional constipation symptoms. (created by figdraw.com ). 5-HT, 5-hydroxytryptamine; SP, substance P; VIP, vasoactive intestinal peptide; SERT, serotonin transporter; EC, enterochromaffin cell.

Article Snippet: Protein A magnetic beads (cat. no. HY-K0203, MedChemExpress) were pre-washed three times with binding/wash buffer provided in the kit and incubated with 10 μ g of anti-Piezo1 antibody (rabbit polyclonal; cat. no. 15939-1-AP; Proteintech Group, Inc.) at 4°C for 2 h to form antibody-bead complexes.

Techniques: Functional Assay, Expressing, Activation Assay, Activity Assay

Journal: Blood Cancer Journal

Article Title: Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia

doi: 10.1038/s41408-022-00660-2

Figure Lengend Snippet: A Schematic representation and surface expression of the CD33-directed second-generation CAR used in this study. Expression was analyzed by flow cytometry 12 days after transgene transfer into primary NK cells. B Time-lapsed expansion of CAR-transduced (CD33-CAR) and untransduced (UTD)-NK cells in the presence of IL-2 (500 IU/mL) and IL-15 (140 IU/mL) ( n = 5). C Expanded NK cells show high cytotoxic activity against various AML cell lines except OCI-AML2. On day 14 of expansion, NK cells were co-incubated with various AML target cells at indicated E:T-ratios. After 24 h, the fraction of viable target cells was quantified by flow cytometry. Data shown are representative of results from two independent experiments. D The AML cell line OCI-AML2 displays high CD33 surface expression. E , F NK cells equipped with a CD33-CAR become highly cytotoxic against OCI-AML2 and CD33-positive primary AML cells. Cells were co-cultivated for 4 h and the viability of target cells was quantitated by flow cytometry. Two representative experiments are shown. G Dynamic monitoring of CAR-NK cell-mediated cytotoxicity. On day 12 after transduction, CAR-NK cells were co-cultured with (GFP + ) OCI-AML2 cells and fluorescence emission was measured in the IncuCyte S3 imaging platform over 4 days. Shown is one representative from three separate experiments with a total of 5 donors. H Repetitive tumor-challenge assay revealed superior serial killing capacity of CD33-CAR-NK cells compared to UTD-NK cells. Expanded NK cells at day 12 post transduction were co-cultured with OCI-AML2 cells at an E:T-ratio of 1:1 and re-challenged with AML cells every other day. Shown is one representative experiment with a total of two donors. All graphs show mean of replicated ± SD.

Article Snippet: An anti-CD56-BV786 antibody (Clone NCAM16.2, BD Biosciences) and a CD33-CAR Detection Reagent (containing a recombinantly expressed fusion protein consisting of the human CD33 extracellular domains and a specifically mutated human IgG1 Fc region, Miltenyi Biotec) followed by an anti-biotin-PE antibody (clone REA746, Miltenyi Biotec) were used to determine CD56 and CD33-CAR expression, respectively.

Techniques: Expressing, Flow Cytometry, Activity Assay, Incubation, Transduction, Cell Culture, Fluorescence, Imaging

Journal: Blood Cancer Journal

Article Title: Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia

doi: 10.1038/s41408-022-00660-2

Figure Lengend Snippet: A Scheme of the in vivo evaluation of a single treatment with CD33-CAR-NK cells (1 × 10 7 intravenously) followed by subcutaneous treatment with IL-2 in OCI-AML2 (Luc + ) xenograft NSG-SGM3 mice. B Total flux analysis as well as representative BLI images of differently treated OCI-AML2 (Luc + ) engrafted NSG-SGM3 mice over time (d7 n = 7; d14 n = 6; d21 n = 5 per group). Mice received a single dose of 1 × 10 7 NK cells day 3 post AML cell injection. At day 21, 4 out of 5 mice (80%) that were treated with CD33-CAR-NK cells show severely reduced leukemic burden compared to untreated mice (UT) or mice which received untransduced (UTD)-NK cells. C Serum analysis of blood day 3 before AML injection and day 1 post first NK cell application shows significantly increased levels of GM-CSF as well as INF-γ for mice that had received CD33-CAR-NK cells ( n = 3). Mean ± SD. D Total flux analysis of femurs/tibiae and spleens, as well as flow cytometry analysis of isolated cells from BMs or spleens at day 7, 14, and 21 post tumor cell injection, revealed the absence of GFP-positive tumor cells in CD33-CAR-NK-treated mice as well as increased NK cell infiltration (day 7/14 n = 1; day 21 n = 2 per group). Values of zero were set to 1 for total flux analysis. Median ± range. Flow cytometry-based CAR expression analysis of BM- ( E ) or spleen- ( F ) infiltrating NK cells at day 14 and 21 revealed the presence of mainly CAR-positive cells (day 14 n = 1; day 21 n = 2 per group). Mean ± SD. G Confocal microscopy imaging shows GFP-positive leukemia cells in BM of UTD-NK treated NSG-SGM3 mice at day 21 while absent in mice that received CD33-CAR-NK cells. Images from one representative animal are shown. Statistical analysis was performed by Student’s t test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: An anti-CD56-BV786 antibody (Clone NCAM16.2, BD Biosciences) and a CD33-CAR Detection Reagent (containing a recombinantly expressed fusion protein consisting of the human CD33 extracellular domains and a specifically mutated human IgG1 Fc region, Miltenyi Biotec) followed by an anti-biotin-PE antibody (clone REA746, Miltenyi Biotec) were used to determine CD56 and CD33-CAR expression, respectively.

Techniques: In Vivo, Injection, Flow Cytometry, Isolation, Expressing, Confocal Microscopy, Imaging

Journal: Blood Cancer Journal

Article Title: Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia

doi: 10.1038/s41408-022-00660-2

Figure Lengend Snippet: A Scheme of the in vivo evaluation of a repetitive treatment with CD33-CAR-NK cells (1 × 10 7 intravenously) combined with subcutaneous IL-2 treatment in OCI-AML2 (Luc + ) xenograft NSG-SGM3 mice. B Total flux analysis, as well as representative BLI images of differently treated OCI-AML2 (Luc+), engrafted NSG-SGM3 mice over time ( n = 7 per group). Mice received a total of three weekly doses of 1 × 10 7 NK cells. Mice that were treated with CD33-CAR-NK cells show severely reduced leukemic burden compared to untreated mice (UT) or mice which received untransduced (UTD)-NK cells. C Total flux analysis of femurs/tibiae and spleens, as well as flow cytometry analysis of isolated cells from BMs or spleens at day 22 post AML-injection, revealed the absence of GFP-positive leukemic cells in CD33-CAR-NK treated mice as well as increased NK cell infiltration ( n = 6–7 per group). Values of zero were set to 1 for total flux analysis. Median ± range. D Chimerism analysis d22 post AML-injection revealed high amounts of DNA from human NK cells without detectable DNA of AML in blood of mice that were treated with CD33-CAR-NK cells ( n = 6–7 per group). Mean ± SD. E Serum analysis of blood day 3 before AML injection and day 1 post first NK cell application showed significantly increased pro-inflammatory human cytokines for mice that received CD33-CAR-NK cells ( n = 6–7 per group). F Flow cytometry-based CAR-expression analysis of BM- or spleen-infiltrating NK cells in CD33-CAR-NK treated mice revealed the presence of mainly CAR-positive cells ( n = 6–7 per group). Mean ± SD. G Confocal microscopy imaging demonstrated GFP-positive leukemia cells in BM of UTD-NK treated NSG-SGM3 mice while absent in mice that received CD33-CAR-NK cells. Additionally, CAR-NK cells could be detected in the BM of CD33-CAR-NK treated mice. One representative image from a total of four are shown. Statistical analysis was performed by Mann–Whitney-test (for total flux analysis) or Student’s t test (for the rest) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: An anti-CD56-BV786 antibody (Clone NCAM16.2, BD Biosciences) and a CD33-CAR Detection Reagent (containing a recombinantly expressed fusion protein consisting of the human CD33 extracellular domains and a specifically mutated human IgG1 Fc region, Miltenyi Biotec) followed by an anti-biotin-PE antibody (clone REA746, Miltenyi Biotec) were used to determine CD56 and CD33-CAR expression, respectively.

Techniques: In Vivo, Flow Cytometry, Isolation, Injection, Expressing, Confocal Microscopy, Imaging, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane

doi: 10.1038/s41419-018-0312-8

Figure Lengend Snippet: a Live measurement of the effect of PXA on uncoupled cellular respiration in intact Ramos cells. Cells were first treated with 1 µM CCCP to uncouple respiration and then with either 10 µM PXA or 0.1% v/v DMSO (vehicle control) to test for inhibition. Measurement of extracellular [O 2 ] was performed using an oxygraph. b Comparative measurements of the effect of PXA and other compounds on basal and uncoupled respiration in Ramos cells. Respiration was uncoupled by CCCP (1 µM). PXA (10 µM) was compared to the complex-specific ETC inhibitors rotenone (Rot; complex I; 10 µM), thenoyltrifluoroacetone (TTFA; complex II; 10 µM), antimycin A (AmA; complex III; 10 µM), azide (NaN 3 ; complex IV; 1 mM) and oligomycin A (OmA; complex V; 10 µM). Measurement was performed in a microplate reader using the fluorescence-based MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) (Enzo). c Comparative measurements of the effect of PXA and other compounds on ATP levels in Ramos cells after 120 min of treatment. PXA (10 µM) was compared to complex-specific ETC inhibitors (see above) as well as CCCP (1 µM). Measurement was performed in a microplate reader using the luminescence-based Mitochondrial ToxGlo™ Assay (Promega). Cells were incubated in full growth medium containing either glucose or galactose as the only available sugar. Galactose alone forces the cells to resort exclusively to OXPHOS for ATP synthesis. d Live measurement of mitochondrial respiration in Ramos cells permeabilized by digitonin (Digi; 5 µg/ml). The effect of PXA (first 1 µM, then increased to 10 µM as indicated) was compared to that of the known complex I inhibitor rotenone (Rot; 1 µM). To specifically induce complex II and III of the electron transport chain, succinate (Succ; 10 mM) and duroquinol (Duro; 1 mM) were used, respectively. Measurement of extracellular [O 2 ] was performed using an oxygraph

Article Snippet: Measurement was performed in a microplate reader using the fluorescence-based MITO-ID® Extracellular O2 Sensor Kit (High Sensitivity) (Enzo). c Comparative measurements of the effect of PXA and other compounds on ATP levels in Ramos cells after 120 min of treatment.

Techniques: Inhibition, Fluorescence, Incubation

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: List of ELISA tests, cell proliferation, and protein extraction kits used. For ELISA tests, dilution of the supernatants or cell lysate samples used and detection limits are also reported.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Enzyme-linked Immunosorbent Assay, Protein Extraction

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: C-Kit (CD117) mRNA (a, b, c) and protein (d, e, f) expression after ESW treatment in primary bronchial fibroblasts of COPD patients (a, d), primary bronchial fibroblasts of control smokers (b, e), and bronchial epithelial cells (c, f). In bronchial epithelium (16HBE) c-Kit increased at mRNA (c) and protein (f) levels. In primary bronchial fibroblasts of COPD patients, c-Kit increased at protein level (d). T -test was used for comparative purposes, and p values are reported in the graphs.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Expressing

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: Photomicrographs showing thyroid transcription factor-1 (TTF-1) expression (panels a, b), c-Kit (CD117) (c, d), and proliferating cell nuclear antigen (PCNA) (e, f) in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary disease (COPD). Arrows indicate positively stained cells mainly located in the alveolar septa. Bars = 50 microns.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Expressing, Staining

Journal: Canadian Respiratory Journal

Article Title: Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients

doi: 10.1155/2020/1524716

Figure Lengend Snippet: Photomicrographs showing alveolar type II epithelial cells (TTF-1+ cells, red color) coexpressing c-Kit (CD117) (brown color) (a, b) and PCNA (brown color) (c, d) in the peripheral lung tissue of a representative patient with COPD. Positive double-stained cells can be recognized in the alveolar septa, even though their presence was only rarely observed. Arrows indicate positively stained cells located in the alveolar septa. Bars = 50 microns.

Article Snippet: c-Kit or SCFR (CD117) , Cloud-Clone Corp. , SEA121 Hu , 1 : 5 (PBS) , 0.61 ng/mL (1.56–100 ng/mL).

Techniques: Staining