Journal: Open Medicine

Article Title: High STAT4 expression correlates with poor prognosis in acute myeloid leukemia and facilitates disease progression by upregulating VEGFA expression

doi: 10.1515/med-2023-0840

Figure Lengend Snippet: SiSTAT4 decreased STAT4 expression, while overexpressed STAT4 did conversely in transfected HL60 and NB4 cells. (a) The expression of STAT4 in HL60 cells after transfection was examined by qPCR, and GAPDH was used as the loading gene. (b) The expression of STAT4 in HL60 cells after transfection was examined by Western blot, and GAPDH was adopted as the internal control. (c) The expression of STAT4 in NB4 cells after transfection was examined by qPCR, and GAPDH was employed as a loading gene. (d) The expression of STAT4 in NB4 cells after transfection was examined by Western blot, and GAPDH was utilized as the internal control. (e) The expression of STAT4 in HL60 cells after transfection was examined by qPCR, and GAPDH was harnessed as the reference gene. (f) The expression of STAT4 in HL60 cells after transfection was measured by Western blot, and GAPDH was exploited as the internal control. (g) The expression of STAT4 in NB4 cells after transfection was quantified by qPCR, and GAPDH was applied as the reference gene. (h) The expression of STAT4 in NB4 cells after transfection was determined by Western blot, and GAPDH was adopted as the loading control. *** P < 0.001 vs siNC; ^^^ P < 0.001 vs NC (NC: negative control, siSTAT4: siRNA targeting STAT4, siNC: negative control of siSTAT4).

Article Snippet: Human AML cell lines including HL60 (CL-0110), KG1 (CL-0132), Kasumi-1 (CL-0556), and NB4 (CL-0676) were purchased from Procell (Wuhan, China), and normal bone marrow cell line HS-5 (CRL-11882) was ordered from American Type Culture Collection (ATCC; Maryland, USA).

Techniques: Expressing, Transfection, Western Blot, Negative Control

Journal: Open Medicine

Article Title: High STAT4 expression correlates with poor prognosis in acute myeloid leukemia and facilitates disease progression by upregulating VEGFA expression

doi: 10.1515/med-2023-0840

Figure Lengend Snippet: STAT4 silencing decreased the viability, while overexpressed STAT4 did conversely in transfected AML cells. (a–d) The viability of HL60 and NB4 cells after transfection was examined by MTT assay. * P < 0.05, *** P < 0.001 vs siNC; ^ P < 0.05, ^^^ P < 0.001 vs NC.

Article Snippet: Human AML cell lines including HL60 (CL-0110), KG1 (CL-0132), Kasumi-1 (CL-0556), and NB4 (CL-0676) were purchased from Procell (Wuhan, China), and normal bone marrow cell line HS-5 (CRL-11882) was ordered from American Type Culture Collection (ATCC; Maryland, USA).

Techniques: Transfection, MTT Assay

Journal: Open Medicine

Article Title: High STAT4 expression correlates with poor prognosis in acute myeloid leukemia and facilitates disease progression by upregulating VEGFA expression

doi: 10.1515/med-2023-0840

Figure Lengend Snippet: STAT4 silencing decreased Bcl-2 level, yet increased apoptosis and Bax level in AML cells, while overexpressed STAT4 did conversely. (a and b) The apoptosis of HL60 and NB4 cells after transfection was examined by flow cytometry. (c) The expressions of Bcl-2 and Bax in HL60 cells after transfection were quantified by qPCR, and GAPDH was used as the reference gene. (d) The expressions of Bcl-2 and Bax in HL60 cells after transfection were detected by Western blot, and GAPDH was used as the loading control. (e) The expressions of Bcl-2 and Bax in NB4 cells after transfection were measured by qPCR, and GAPDH was used as the reference gene. (f) The expressions of Bcl-2 and Bax in NB4 cells after transfection were examined by Western blot, and GAPDH was used as the internal control. *** P < 0.001 vs siNC; ^^^ P < P < 0.001 vs NC.

Article Snippet: Human AML cell lines including HL60 (CL-0110), KG1 (CL-0132), Kasumi-1 (CL-0556), and NB4 (CL-0676) were purchased from Procell (Wuhan, China), and normal bone marrow cell line HS-5 (CRL-11882) was ordered from American Type Culture Collection (ATCC; Maryland, USA).

Techniques: Transfection, Flow Cytometry, Western Blot

Journal: Open Medicine

Article Title: High STAT4 expression correlates with poor prognosis in acute myeloid leukemia and facilitates disease progression by upregulating VEGFA expression

doi: 10.1515/med-2023-0840

Figure Lengend Snippet: STAT4 silencing decreased the angiogenesis and VEGFA level, while overexpressed STAT4 did conversely. (a and b) The angiogenesis of HL60 and NB4 cells after transfection was examined by tube formation assay under ×100 magnification. (c) The expressions of STAT4 and VEGFA in HL60 cells after transfection were tested by qPCR, and GAPDH was used as the reference gene. (d) The expressions of STAT4 and VEGFA in HL60 cells after transfection were determined by Western blot, and GAPDH was adopted as the internal control. (e) The expressions of STAT4 and VEGFA in NB4 cells after transfection were assayed by qPCR, and GAPDH was utilized as the reference gene. (f) The expressions of STAT4 and VEGFA in NB4 cells after transfection were measured by Western blot, and GAPDH was exploited as the loading control. *** P < 0.001 vs siNC; ^^^ P < 0.001 vs NC (VEGFA: vascular endothelial growth factor A).

Article Snippet: Human AML cell lines including HL60 (CL-0110), KG1 (CL-0132), Kasumi-1 (CL-0556), and NB4 (CL-0676) were purchased from Procell (Wuhan, China), and normal bone marrow cell line HS-5 (CRL-11882) was ordered from American Type Culture Collection (ATCC; Maryland, USA).

Techniques: Transfection, Tube Formation Assay, Western Blot

Journal: Open Medicine

Article Title: High STAT4 expression correlates with poor prognosis in acute myeloid leukemia and facilitates disease progression by upregulating VEGFA expression

doi: 10.1515/med-2023-0840

Figure Lengend Snippet: VEGFA silencing counteracted the effects of overexpressed STAT4 on promoting the viability and angiogenesis as well as inhibiting the apoptosis of AML cells. (a and b) The viability of HL60 and NB4 cells after transfection was examined by MTT assay. (c and d) The angiogenesis of HL60 and NB4 cells after transfection was determined by tube formation assay under ×100 magnification. (e and f) The apoptosis of HL60 and NB4 cells after transfection was tested by flow cytometry. * P < 0.05, ** P < 0.01 , *** P < 0.001 vs NC + vector; ^ P < 0.05, ^^ P < 0.01, ^^^ P < 0.001 vs STAT4 + vector; # P < 0.05, ## P < 0.01, ### P < 0.001 vs NC + siVEGFA (siVEGFA: siRNA targeting VEGFA, vector: negative control for siVEGFA).

Article Snippet: Human AML cell lines including HL60 (CL-0110), KG1 (CL-0132), Kasumi-1 (CL-0556), and NB4 (CL-0676) were purchased from Procell (Wuhan, China), and normal bone marrow cell line HS-5 (CRL-11882) was ordered from American Type Culture Collection (ATCC; Maryland, USA).

Techniques: Transfection, MTT Assay, Tube Formation Assay, Flow Cytometry, Plasmid Preparation, Negative Control

Journal: Cell & Bioscience

Article Title: Transcriptionally imprinted glycomic signatures of acute myeloid leukemia

doi: 10.1186/s13578-023-00981-0

Figure Lengend Snippet: Overview of the conducted study. AML glycosylation was explored on the level of glycomics (GPST datasets) and transcriptomics (GSE and DepMap datasets). Based on the depicted datasets originating from cell lines and primary cells we sought to explore cellular glycosylation, involved GSTs, and responsible TFs

Article Snippet: AML cell line transcriptomics data was obtained from the depmap portal (Expression 22Q2 Public; Broad Institute, Cambridge, MA, USA) [ ].

Techniques: Glycoproteomics

Journal: Cell & Bioscience

Article Title: Transcriptionally imprinted glycomic signatures of acute myeloid leukemia

doi: 10.1186/s13578-023-00981-0

Figure Lengend Snippet: Glycomic overview of various AML cell lines. a PCA of glycosylation features derived from glycomics data of 19 AML cell lines. Individual cell lines are annotated and colored by their FAB classifications as assigned earlier . b The associated score plot depicts considered glycan features, which are linked to their respective glycan class ( N -, O -, and GSL) by color (purple, orange, and green) and symbol (triangle, square, and circle). In addition, arrows indicate features that are linked to a specific type of fucosylation. c Radar plots are showing the differences in glycosylation features between AML classes M5 and M6. Again, these features are subdivided into their respective classes based on color and symbols. Data on all AML cell lines were z-transformed prior to visualizing differences between FAB classes in these radar plots. d Spearman correlation of selected glycosylation features between the different glycan classes. Thick connective lines indicate a good correlation whereas thin connective lines show less correlation. Correlation values are depicted

Article Snippet: AML cell line transcriptomics data was obtained from the depmap portal (Expression 22Q2 Public; Broad Institute, Cambridge, MA, USA) [ ].

Techniques: Glycoproteomics, Derivative Assay, Transformation Assay

Journal: Cell & Bioscience

Article Title: Transcriptionally imprinted glycomic signatures of acute myeloid leukemia

doi: 10.1186/s13578-023-00981-0

Figure Lengend Snippet: Correlation of glycosylation features of N -, O -, and GSL-glycans with the expression of selected TFs in AML cell lines. Correlation coefficients were obtained by Spearman analysis and are indicated by color as indicated in the legend. Of note, due to rather weak correlations of ST6GALs and glycomics data, we did not include these GSTs in our overview. Significant values are marked with * (p ≤ 0.05), ** (p ≤ 0.01,) and *** (p ≤ 0.001). Correlation coefficients and p-values are listed in the Additional file : Table S9

Article Snippet: AML cell line transcriptomics data was obtained from the depmap portal (Expression 22Q2 Public; Broad Institute, Cambridge, MA, USA) [ ].

Techniques: Glycoproteomics, Expressing

Journal: Cell & Bioscience

Article Title: Transcriptionally imprinted glycomic signatures of acute myeloid leukemia

doi: 10.1186/s13578-023-00981-0

Figure Lengend Snippet: Differences in glycan signatures of M5 and M6 AML cell lines as well as corresponding GST and TF expression. M5 and M6 classes are presented as grey and brown rectangles, respectively. GSTs displayed in the figure present a positive correlation with the corresponding glycosylation feature. The underlined TFs correlate with the glycosylation features. The underlined TFs colored in red are positively correlated with GSTs

Article Snippet: AML cell line transcriptomics data was obtained from the depmap portal (Expression 22Q2 Public; Broad Institute, Cambridge, MA, USA) [ ].

Techniques: Glycoproteomics, Expressing

Journal: Cell & Bioscience

Article Title: Transcriptionally imprinted glycomic signatures of acute myeloid leukemia

doi: 10.1186/s13578-023-00981-0

Figure Lengend Snippet: GST and TF expression in primary AML cells. a Determination of the matrix correlation coefficient RV2 (0.49) between expression patterns observed in cell lines and primary samples. b Spearman correlation of selected GSTs with TFs in AML cell lines (left) and primary AML cells (right). c Comparison of the expression of selected GSTs and TFs in primary AML cells grouped by FAB classification. Significances were assessed by one-way ANOVA followed by a Tukey post-hoc test. Significant values are marked with * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), and **** (p ≤ 0.0001)

Article Snippet: AML cell line transcriptomics data was obtained from the depmap portal (Expression 22Q2 Public; Broad Institute, Cambridge, MA, USA) [ ].

Techniques: Expressing, Comparison

Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: Inhibition of AMPK activity by venetoclax in AML cells. ( A ) Four different AML cell lines were seeded in 384-well plates at 3 × 10 5 cells/mL and incubated with a dose range of venetoclax during 24 h. Cell viability was then measured using the ATPlite luminescent reagent, and results were analyzed with the nonlinear regression module and plotted using log(inhibitor) versus response (three parameters) function of the Prism software. ( B ) Four different AML cell lines were seeded in 384-well plates at 3 × 10 5 cells/mL and incubated with 100 nM venetoclax during 0 h o 8 h. Then, ATP content was measured using the ATP lite luminescent reagent. Results of ATP content (Y-axis) were plotted on incubation time (X-axis) and a linear regression was performed using the Prism software. Time for achieving a 50% reduction of ATP content is indicated by vertical dashed lines for each cell lines following their respective color code. ( C ) AML cell lines were seeded at 5 × 10 5 cells/mL and incubated with 100 nM venetoclax for the indicated times. Western blots were performed using the anti-phospho-AMPKα T172, -AMPKα, -Bcl-2, and -β-actin antibodies. ( D ) AML cell lines were incubated with vehicle (DMSO) or 100 nM venetoclax (VEN) for 6 h, and Western blots were performed using anti-phospho-ACC S79, -phospho-ULK-1 S555, -phospho-AMPKα T172, -AMPKα, and -β-actin antibodies.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Inhibition, Activity Assay, Incubation, Software, Western Blot

Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: Decreased amount of AMPK subunits by venetoclax in AML. ( A ) Western blots were done from in vitro kinase assay protein mix using anti-phospho-ACC S79 and anti-phospho-AMPK T172 antibodies. ( B , C ) AML cell lines were incubated without or with 100 nM venetoclax (VEN), and then submitted to a cycloheximide pulse during the indicated times. ( B ) Western blots were done using anti-AMPKα, β, γ, -Bcl-2, and -β-actin antibodies. ( C ) Quantification of the Western blot signals from three independent experiments using ImaJ software for AMPKα detection in the control (CTR) or venetoclax (VEN) conditions.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Western Blot, In Vitro, Kinase Assay, Incubation, Software, Control

Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: AMPK degradation is due to on-target caspase activation by venetoclax. ( A ) AML cell lines were incubated with 100 nM venetoclax during the indicated times and processed for flow cytometry using annexin V and DAPI staining. Annexin V-positive and DAPI-negative cells are those in early apoptosis, while double positivity indicates either late apoptosis or necrotic cells. The experiment was repeated three times separately. Vertical bars indicate standard deviations. * p < 0.05, *** p < 0.001. ( B , C ) AML cell lines were incubated with vehicle (CTR), 100 nM venetoclax (VEN), 50 µM Z-VAD (pan-caspase inhibitor) or a combination of 50 µM Z-VAD (added 24 h before VEN) and 100 nM venetoclax for 4 h. ( B ) Flow cytometry measurement of annexin V binding done in three separate experiments and plotted in a heat-map format. ( C ) Western blots done using anti-phospho-AMPK T172, -AMPK α, β, γ, -cleaved caspase 3, and –β-actin.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Activation Assay, Incubation, Flow Cytometry, Staining, Binding Assay, Western Blot