Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Western bloting analysis of total PCLP1 expression in B cell lines (Raji, Ramos and Daudi), B cells from four healthy donors (B cells 1–4), and a T cell line (Jurkat). Actin is shown as a loading control. ( B ) PCLP1 expression on the surface of various B cell lines and Jurkat T cell line by flow cytometry. The grey line and the red line in the histograms represent isotype control (mouse IgG) and PCLP1 staining (anti-PCLP1 mAb), respectively. ( C ) Raji cells were stably transfected with pEGFP-PCLP1 (Raji-PCLP1) or pEGFP (Raji-Ctrl) and whole cell extracts from three different clones of each cell type were analyzed for the expression of PCLP1 by Western-blotting using an anti-hPCLP1 mAb. PCLP1: endogenous PCLP1. PCLP1-GFP: ectopic PCLP1. Actin is shown as a loading control. ( D ) Representative fluorescence microscope images of Raji cells stably expressing PCLP1-GFP fusion protein showing the localization of PCLP1-GFP (green) to cell surface and vesicles (I), and centrosome (II), using a 60× objective. Nuclei were stained with Hoechst 33342 (blue). ( E ) Representative fluorescence microscope images of Raji-PCLP1 cells showing the centrosomal localization of PCLP1-GFP fusion protein (green) at interphase (top) and mitosis (bottom). Cells were stained with an anti-γ-tubulin mAb to visualize the pericentriolar area of the centrosome (red). Nuclei were stained with DAPI (blue). Insets at lower right corner show amplified images of the centrosomal region. The size bar indicates 5 µm. ( F ) PCLP1 expression was determined on the surface of malignant and normal B cells from 11 patients with different lymphoma subtypes and 3 patients with ALL by flow cytometry. Median fluorescence intensity (MFI) of PCLP1 expression on malignant B cells was normalized to that detected on normal B cells from the same patient. N: normal B cells; M: malignant B cells; B-ALL: B-cell acute lymphoblastic leukemia; FL: follicular lymphoma; WM: Waldenström macroglobulinemia; HCL: hairy cell leukemia; CLL: chronic lymphocytic leukemia.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Staining, Stable Transfection, Transfection, Clone Assay, Fluorescence, Microscopy, Amplification

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Cell proliferation of Raji-PCLP1 and Raji-Ctrl cell clones was measured counting the number of viable cells at different time points of culture in complete medium using the trypan blue exclusion assay. ( B ) Colony formation of Raji-PCLP1 and Raji-Ctrl cells cultured in methylcellulose medium for 11 days. Colonies were stain with crystal violet and counted using an inverted microscope. The graph depicts the number of colonies per well from four independent experiments performed in triplicate. Representative microscope pictures and enlarged images of colony formation after crystal violet staining are shown. Statistical analysis of mean ± SD was calculated using two-tailed paired Student´s t -test. *** P < 0.001. ( C ) Cell-to-cell adhesion was calculated as the number of cell aggregates formed at 72 h of culture. Representative microscope images of cell aggregates using a 10× objective are shown. The size bar indicates 100 µm. ( D ) Cells were incubated in the presence of anti-CD62L mAb or isotype control (mIgG1) (left) or in the presence of anti-CD18 mAb or mIgG1 (right) and the number of aggregates were counted at 2 h. Cells were cultured in the presence of 1 mM EDTA, 1 mM EGTA or vehicle and the number of aggregates were counted at 24 h (middle). The graphs in C and D represents the mean ± SD of the number of aggregates per field containing more than 20 cells from 3 independent experiment and analyzing 10 fields in each experiment. Statistical analysis was calculated using no-paired Student´s t -test (C) and Mann-Whitney U test (D). ** P < 0.01 *** P < 0.001. ( E ) Chemotactic migration of Raji-PCLP1 or Raji-Ctrl toward 100 ng/ml CXCL12 using Transwell chambers. Data represents the mean chemotactic index ± SD normalized to control cells from six independent experiments. Statistical analysis was calculated using Mann-Whitney U test. ** P < 0.01.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Clone Assay, Trypan Blue Exclusion Assay, Cell Culture, Staining, Inverted Microscopy, Microscopy, Two Tailed Test, Incubation, Control, MANN-WHITNEY, Migration

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Raji-Ctrl and Raji-PCLP1 cells were treated with increasing concentrations of vehicle or dexamethasone for 72 hours and cell death was analyzed after Annexin V-PE/7´AAD staining by flow cytometry. The graph on the left shows the mean percentages ± SD of total specific cell death (including early apoptosis, late apoptosis and necrotic cells) from six independent experiments after subtracting the spontaneous cell death (vehicle). The percentage of specific cell death was calculated as follows: [(% lysis of target cell – % spontaneous cell death)/(100% – % spontaneous cell death)] × 100. Representative dot plots showing Annexin V/7AAD staining in cells treated with vehicle or 1000 µM dexamethasone are shown (right). As displayed in the dot plots, the spontaneous cell death (Annexin V PE positive/7ADD negative, Annexin V PE positive/7ADD positive and Annexin V PE negative/7ADD positive cells) in cells cultured with the vehicle was approximately 7.5% in Raji-Ctrl and 5.5% in Raji-PCLP1 cells. ( B ) Expression of PCLP1 on Annexin V and 7ADD double-negative population of cells treated with dexamethasone or vehicle by flow cytometry. ( C ) Raji-Ctrl and Raji-PCLP1 cells were treated with increasing concentrations of vehicle or H 2 O 2 for 6 h and cell death was analyzed as indicated in A. The graph on the left shows the mean percentages ± SD of total cell death from five independent experiments after subtracting the spontaneous cell death (vehicle). Representative dot plots showing Annexin V/7AAD staining in cells treated with vehicle or 200 µM H 2 O 2 are shown (left). As displayed in the dot plots, the spontaneous cell death in the presence of vehicle was approximately 17.0% in Raji-Ctrl and 5.9% in Raji-PCLP1 cells. ( D ) Cells were incubated overnight with increasing concentrations of obinutuzumab or vehicle and cell viability was measured based on Annexin V/7AAD double negative staining relative to vehicle-treated cells. Data show the mean percentages ± SD of viable cells from six independent experiments. ( E ) A representative image of the homotypic adhesion induced by obinutuzumab as visualized by light microscopy is shown. The size bar indicates 100 µm. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Staining, Flow Cytometry, Lysis, Cell Culture, Expressing, Incubation, Negative Staining, Light Microscopy, Two Tailed Test

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Measurement of pH of Raji-PCLP1 or Raji-Ctrl cell culture medium containing glutamine (complete medium) (left) or in the absence of glutamine (right) at the indicated time points. ( B ) Proliferation of Raji-PCLP1 and Raji-Ctrl cells in glucose-free culture medium (left) or glutamine-free culture medium (middle) at different time points. The number of viable cells was normalized to their counterparts grown in complete medium. Data show the mean ± SD of five independent experiments. Proliferation of Raji-PCLP1 and Raji-Ctrl cells in glucose-free conditions and decreasing concentrations of glutamine at 72 h of culture (right). The number of viable cells was normalized to their counterparts grown in complete medium. Data show the mean ± SD of six independent experiments. ( C ) Death of Raji-PCLP1 and Raji-Ctrl cells under glutamine-deprivation conditions. Cells were incubated in the presence of glucose and decreasing concentration of glutamine and the percentage of cell death was determined at 72 h of culture after Annexin V-PE/7´AAD staining by flow cytometry. Data show the mean ± SD of five independent experiments. Representative dot plots showing Annexin V/7AAD staining in cells treated with complete medium (2 mM glutamine/11 mM glucose) or glutamine free medium (0 mM glutamine/11 mM glucose) are depicted. ( D ) Effect of glucose depletion on PCLP1 expression in Raji cells. Cells were cultured in high-glucose medium (11 mM) or low-glucose medium (0.5 mM) for 72 h and the expression of PCLP1 on cell surface was determined by flow cytometry. Data show MFI of PCLP1 expression from five independent experiments. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Cell Culture, Incubation, Concentration Assay, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: Determination of lipid droplet content in Raji-PCLP1 and Raji-Ctrl cells ( A ) or Jurkat-PCLP1 and Jurkat-Ctrl cells ( B ) by fluorescence microscopy. Cells were cultured in complete medium for the indicated times, fixed and stained with Nile Red for the detection of neutral lipid droplets. Ten fields were randomly selected using a 60× oil objective and the total area of lipid droplets per cell was calculated employing ImageJ software. Bar graphs represent the quantitative analysis of lipid droplets content per cell. Mean ± SD of lipid droplet area per cell is shown. Statistical analysis was calculated using Mann-Whitney U test. * P < 0.05. ** P < 0.01. *** P < 0.001. Representative fluorescence microscopy images depicting lipid droplets stained with Nile Red (right panels, yellow). Nuclei were labeled with DAPI (left panels, blue). Digital zoom of a selected area is shown. The size bar indicates 10 µm.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Fluorescence, Microscopy, Cell Culture, Staining, Software, MANN-WHITNEY, Labeling

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Schematic representation of cancer cell metabolism. Once transported into cells, glucose is phosphorylated to glucose 6-phosphate by hexokinases. Glucose 6-phosphate can then be metabolized via aerobic glycolysis or PPP. Through the aerobic glycolysis, glucose 6-phosphate is sequentially converted to pyruvate. Afterwards, pyruvate is transformed to acetyl-CoA via TCA or to lactate. Acetyl-CoA can be used for the synthesis of fatty acids and lipids for biomembrane generation. Glucose 6-phosphate that enters PPP generates ribose, for nucleotides synthesis, and NADPH, for FA synthesis. Glutamine provides intermediates to maintain TCA function and contributes to lipid synthesis and the production of purines and pyrimidines for nucleotide synthesis. Glucose and glutamine also provide the carbon and nitrogen necessary for the synthesis of nonessential amino acids. Metabolic inhibitors are depicted in red color. 2DG: 2-deoxyglucose; 6AN: 6 aminonicotinamide. ( B ) Raji-PCLP1 and Raji-Ctrl cells were cultured for 72 h in complete medium in the absence or in the presence of increasing concentrations of the 2-DG (glycolysis inhibitor). ( C ) To determine the effect of compound 968 (glutaminase inhibitor) on cell proliferation, Raji-PCLP1 and Raji-Ctrl cells were cultured for 7 days in complete medium in the absence or in the presence of increasing concentrations of the inhibitor. ( D ) Raji-PCLP1 and Raji-Ctrl cell were cultured for 72 h in complete medium in the absence or in the presence of increasing concentrations of cerulenin (fatty acid synthase inhibitor). ( E ) Raji-PCLP1 and Raji-Ctrl cells were cultured for 72 h in complete medium in the absence or in the presence increasing concentrations of 6AN (PPP inhibitor). Cell proliferation was assessed by counting live cells on a hemocytometer using trypan blue exclusion dye. The number of viable cells was normalized to their counterparts grown in the absence of the inhibitor. Data show the mean ± SD percentage relative to non-treated cells of five independent experiments. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Transformation Assay, Cell Culture, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

doi: 10.1038/cddis.2015.410

Figure Lengend Snippet: Involvement of mTOR in GPAT1-induced mESCs anti-apoptosis under hypoxia. ( a ) Cells were transfected with gpat1 and NT siRNA for 24 h before glucosamine (10 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Collected samples are lysed, and p-mTOR, GPAT1, and β -actin protein expressions were measured by using western blotting. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with glucosamine. ( b ) Cells were pretreated with rapamycin (10 nM) before glucosamine (10 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, and β -actin. Each result shown is representative of three independent experiments. ( c ) Cells were pretreated with glucosamine and/or rapamycin (10 nM) for 30 min before hypoxia treatment, and cytochrome c, COX IV, and β -tubulin in the cytosolic and mitochondrial fraction were detected by western blot. ( d ) Cells were pretreated with various concentrations of LPA (10 − 6 M–10 − 9 M) before hypoxia treatment for 24 h. Cell viability was measured by trypan blue exclusion assay. Error bars are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( e ) Cells were pretreated with pertussis toxin (100 ng/ml) for 30 min before LPA treatment (0.1 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Total proteins were extracted and blotted with p-mTOR, mTOR, p-S6K1, S6K1, p-4EBP1, 4EBP1, and β -actin. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( f ) p-mTOR was immunostained with p-mTOR antibody, and counter-stained with PI. Fluorescence images were acquired by using confocal microscopy. Fluorescence intensity of p-mTOR was quantified by using ImageJ software. Data are presented as a mean±S.E.M. of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( g ) Cells were pretreated with rapamycin (10 nM) before LPA (0.1 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, and β -actin. Each result shown is representative of three independent experiments. ( h ) Cell viability was measured by using cell counter. Data are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( i ) Viable cells were measured by using annexin V/PI flow cytometry analysis. Annexin V-negative-PI-negative cells (Q3) were considered viable, annexin V-negative-PI-positive cells (Q1) were considered necrotic, annexin V-positive-PI-positive cells (Q2) were considered late apoptotic, and annexin V-positive-PI-negative cells (Q4) were considered early apoptotic. Data are presented as a mean±S.E.M. of two independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. * P <0.05 versus control, # indicates P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. The proposed model for signaling pathways involved in glucosamine-induced mESCs survival under hypoxia ( j )

Article Snippet: Mammalian target of rapamycin (mTOR), p-mTOR (Ser 2448), S6K1, p-S6K1 (Thr 389), 4EBP1, and p-4EBP1 (Thr 37/46) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Western Blot, Control, Trypan Blue Exclusion Assay, Staining, Fluorescence, Confocal Microscopy, Software, Flow Cytometry, Protein-Protein interactions

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a Western blotting was used to verify FHIT knockout and overexpression in lung cancer cell pairs. HCC827 and H1650 are FHIT-expressing lines, and H1299 and H460 are FHIT-deficient lines. GAPDH was used as the internal control. b Schematic illustration of highly selective inhibitor library screening to discover synthetic lethal candidates for FHIT deficiency. c The scatter plot presents the screening results as log10-IC 50 values of the inhibitors. Inhibitors with an SI (parental-IC 50 / FHIT −/− -IC 50 ) greater than two were considered synthetic lethal drugs for FHIT defects and are highlighted with colored dots. d – f Cell confluence was acquired from the Incucyte Zoom and normalized to that of the control group to validate the synthetic lethal effects of GSK3β inhibition. HCC827 (parental and FHIT −/− ), H460 ( FHIT OE and parental) and H1299 ( FHIT OE and parental) cell pairs were treated with different doses of CHIR99021 for three days. Representative images of HCC827 ( g ), H460 ( h ) and H1299 ( i ) cells treated with the indicated concentrations of CHIR99021 for three days; scar bar = 50 μm. j Representative images of a colony formation assay in HCC827-FHIT isogenic cells. The cells were treated with 2.5 μM or 10 μM CHIR99021 (CHIR) for 10 days and stained with crystal violet. k Effects of CHIR99021 on the growth rate of HCC827 FHIT-isogenic cell pairs. The cells were incubated with 10 μM CHIR99021, and an IncuCyte Zoom was used to capture images of the cells at different time points to calculate cell confluence. The data are presented as the mean ± SD of each group. The statistical difference between the two groups was analyzed by two-way ANOVA. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Western Blot, Knock-Out, Over Expression, Expressing, Control, Library Screening, Inhibition, Colony Assay, Staining, Incubation

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a Synthetic lethality in FHIT-knockdown HCC827 cells treated with CHIR99021 (CHIR). After HCC827 cells were transfected with or without 200 nM FHIT siRNA for 48 h, they were incubated with CHIR99021 (0, 5, 10, or 20 μM) for an additional three days. Cell viability was assessed via the Alamar blue assay. The data are presented as the mean ± SD of each group. Unpaired two-tailed Student’s t tests were used to determine the significance of the differences between two groups. b Representative cell images of a , scale bar = 50 μm. c Western blot analysis of FHIT protein knockdown in a . d Synthetic lethality in FHIT-knockdown H1650 cells treated with CHIR99021. After H1650 cells were transfected with or without 200 nM FHIT siRNA for 48 h, they were incubated with CHIR99021 (0, 5, 10, or 20 μM) for an additional three days. Cell viability was assessed via the Alamar blue assay. The data are presented as the mean ± SD of each group. Unpaired two-tailed Student’s t tests were used to determine differences between two groups. e Representative cell images of d , scale bar = 50 μm. f Western blot analysis of FHIT protein knockdown in d . g ‒ l Synthetic lethality of GSK3β knockdown in HCC827 FHIT −/− cells. After HCC827 parental and FHIT −/− cells were transfected with or without 100 nM GSK3α ( g ) or GSK3β ( j ) siRNA for 72 h, cell viability was determined via the Alamar blue assay. The data are presented as the mean ± SD of each group. Unpaired two-tailed Student’s t tests were used to determine the significance of the differences between two groups. NS denotes not significant. Representative cell images h , k of g and j , scale bar = 50 μm. Western blotting was used to detect the knockdown of the GSK3α ( i ) or GSK3β ( l ) proteins in g and j . GAPDH was used as the internal control. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Knockdown, Transfection, Incubation, Alamar Blue Assay, Two Tailed Test, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a Western blotting was used to compare the protein levels of GSK3β and p-GSK3α/β (Y279/Y216) in four lung cancer cell lines, FHIT-expressing (H1650 and HCC827) and FHIT-defective (H460 and H1299) lines. b Western blot analysis of the protein levels of GSK3β and p-GSK3α/β (Y279/Y216) in the HCC827 FHIT-isogenic cell pair. c Western blot analysis of γ-H2A. X protein levels in the HCC827 and H1650 FHIT-isogenic cell pairs. d – i Effects of GSK3βi on DNA double-strand breaks in HCC827 and H1650 FHIT isogenic cells. The cells were treated with or without 20 μM CHIR99021 for 24 h, and the protein level of γ-H2A. X was examined by Western blotting ( d , g ), and the in situ foci of γ-H2A. X were detected via immunofluorescence staining, as shown in the fluorescence images; scale bar = 100 μm. Inset images (red squares) are enlarged and shown below each panel; scale bar = 10 μm. The nuclei were stained with DAPI ( e , h ), and the number of cells containing more than five γ-H2A. X foci in each group were recorded and quantified via ImageJ with a threshold (setting a fixed value: 74, 129) to distinguish positive γ-H2A. X foci from the background staining ( f , i ). The data are presented as the mean ± SD of each group. The difference between two groups was determined by an unpaired two-tailed Student’s t test. j , k Comet assay to test DNA damage in cells upon GSK3βi treatment. HCC827 FHIT-isogenic cells were treated with or without 20 μM CHIR99021 for 24 h to conduct an alkaline comet assay, and representative images from the comet assay are shown; scale bar = 200 μm. Inset images (red squares) are enlarged and shown below each panel; scale bar = 50 μm. Representative images ( j ) were acquired via an Evos microscope (Life Technology), and the percentage of DNA in the tail ( k ) was analyzed via the ImageJ component Open Comet. The data are presented as the mean ± SD of each group. The significance of the difference between two groups was determined by an unpaired two-tailed Student’s t test. GAPDH was used as the internal control. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Western Blot, Expressing, In Situ, Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Single Cell Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Microscopy, Control

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a – d Effect of FHIT deficiency on the protein stability of BRCA1 and RAD51. After HCC827 parental and HCC827 FHIT −/− cells were incubated with 50 μg/mL CHX for the indicated times, Western blotting was performed to determine the protein levels of BRCA1 ( a ) and RAD51 ( c ), and ImageJ software was used to quantify the percentage of BRCA1 ( b ) and RAD51 ( d ) protein remaining. e , f HSP90 interacts with FHIT and BRCA1/RAD51. BioID was used to detect the interaction between FHIT and HSP90 in HCC827 cells ( e ), and HSP90 IP was used to analyze the interactions between HSP90 and FHIT, BRCA1, and RAD51 ( f ). g Western blot analysis of the protein levels of BRCA1 and RAD51 in HCC827 FHIT-isogenic cells treated with 125 nM geldanamycin for 48 h. GAPDH was used as the internal control. h – j Effect of a GSK3β inhibitor on the protein stability of BRCA1 and RAD51. After the cells were treated with or without 20 μM CHIR99021 for 30 min and then incubated with 50 μg/mL CHX for the indicated times, Western blotting was performed to determine the protein levels of BRCA1 and RAD51 in HCC827 FHIT −/− cells ( h ), and ImageJ software was used to quantify the percentage of remaining BRCA1 and RAD51 proteins ( i , j ). k Effect of a GSK3β inhibitor on the mRNA levels of BRCA1 and RAD51. After the HCC827 FHIT −/− cells were treated with or without 20 μM CHIR99021 for 12 h, RT‒qPCR was used to examine the mRNA levels of BRCA1 and RAD51. The data are presented as the means ± SDs of each group. The difference between two groups was determined by an unpaired two-tailed Student’s t test. l The effect of si Snail on CHIR99021-induced BRCA1 and RAD51 downregulation. HCC827 FHIT −/− cells were transfected with 200 nM Snail siRNA for 72 hours and then treated with 20 μM CHIR99021 for 24 h. The cell lysates were analyzed via Western blotting for Snail, BRCA1 and RAD51. GAPDH was used as the internal control. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Incubation, Western Blot, Software, Control, Two Tailed Test, Transfection

Journal: Journal of animal science and biotechnology

Article Title: Increased maternal consumption of methionine as its hydroxyl analog improves placental angiogenesis and antioxidative capacity in sows.

doi: 10.1186/s40104-025-01159-z

Figure Lengend Snippet: Fig. 7 Increased consumption of methionine as OHMet improved angiogenesis and antioxidative capacity in pTr cells. A The trophoblast cells were treated for 24 h with 20% serum from each group (n = 3). B Cell viability. C Reactive oxidative species level. D Schematic of the Met metabolism and list of genes directly involved and associated with this pathway. E The gene expression of transmethylation and trans-sulphuration. F The gene expression of anti-oxidation. G The gene expression of angiogenesis. E–G Duplicated independent experiments. H The protein expression of pTr, GAPDH as a loading control. TXN, thioredoxin; GSTT1, glutathione transferase T1; VEGF-A, vascular endothelial growth factor A. Significant difference among four treatments is marked with different superscripts (P < 0.05). Abbreviations: CON = basal diet; 1.5S-OHMet = basal diet + 1.5 g/kg OHMet; 3.0S-OHMet = basal diet + 3.0 g/kg OHMet; 3.0S-Met = basal diet + 3.0 g/kg Met

Article Snippet: Primary antibodies were thioredoxin (TXN, Zen BioScience, 1:1,000), HADHA (Zen BioScience, 1:1,000), NDUFA3 (Zen BioScience, 1:1,000), ENO3 (Zen BioScience, 1:1,000), GSTT1 (Proteintech, 1:1,000), GAPDH (Absin, 1:1,000), β-actin (Cell Signaling Technology, 1:1,000).

Techniques: Gene Expression, Expressing, Control

Journal: Journal of animal science and biotechnology

Article Title: Increased maternal consumption of methionine as its hydroxyl analog improves placental angiogenesis and antioxidative capacity in sows.

doi: 10.1186/s40104-025-01159-z

Figure Lengend Snippet: Fig. 8 Anti-proliferative and inhibited antioxidant capacity induced by homocysteine in pTr cells (n = 3). A Detection of LDH activity in pTr cells. B EdU-positive cells were measured using ImageJ software. C Cell proliferation was determined by the EdU assay, and images were taken under a fluorescence microscope (magnification 400 × , bar = 100 μm). D Cell migration was measured using ImageJ software. E The cell migration distance (bar = 500 μm) and images of ROS were taken using a DCFH-DA probe (magnification 400 × , bar = 500 μm). F Representative western blot results for protein levels of enolase 3 (ENO3) and thioredoxin (TXN), β-actin was used as a loading reference. G–J Detection of apoptosis in pTr cells. Annexin V and propidium iodide (PI) fluorescence was quantified using flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Primary antibodies were thioredoxin (TXN, Zen BioScience, 1:1,000), HADHA (Zen BioScience, 1:1,000), NDUFA3 (Zen BioScience, 1:1,000), ENO3 (Zen BioScience, 1:1,000), GSTT1 (Proteintech, 1:1,000), GAPDH (Absin, 1:1,000), β-actin (Cell Signaling Technology, 1:1,000).

Techniques: Activity Assay, Software, EdU Assay, Fluorescence, Microscopy, Migration, Western Blot, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding