mouse aml cell lines thp 1  (ATCC)

Journal: Translational Oncology

Article Title: REST-driven upregulation of SFXN3 promotes AML progression via Wnt/β-catenin activation and confers decitabine resistance

doi: 10.1016/j.tranon.2026.102705

Figure Lengend Snippet: Effects of SFXN3 Knockdown on Proliferation, Apoptosis, and Signaling Pathways in AML Cells. (A–C) qRT-PCR and Western blot analyses were used to measure SFXN3 expression levels in various leukemia cell lines (THP-1, KG-1, U937, K562) and in normal bone marrow stromal cells (HS-5). (D) Two independent shRNAs (sh-SFXN3–1 and sh-SFXN3–2) were used to knock down SFXN3 expression in THP-1 and KG-1 cells. Western blot was performed to assess the knockdown efficiency and specificity. (E) Quantification of SFXN3 knockdown efficiency by different shRNAs. (F) CCK-8 cell proliferation assays were conducted to evaluate the effects of SFXN3 knockdown on cell growth dynamics over time. (G) EdU incorporation assays were used to assess DNA synthesis activity, indirectly reflecting cellular proliferation, and to compare differences between knockdown and control groups, (bar=50ųm). (H) Western blot analysis of key cell cycle regulatory proteins (CDK4, CDK6, P27, and P21) to investigate the potential mechanism by which SFXN3 affects cell cycle progression. (I) TUNEL assays were used to evaluate apoptosis levels in the knockdown versus control groups, assessing the role of SFXN3 in apoptosis suppression, (bar=50ųm). (J) Western blot analysis of pro-apoptotic proteins (BAX and BAK) and anti-apoptotic proteins (Bcl-2 and Bcl-xl) in THP-1 and KG-1 cells following SFXN3 knockdown. (K) Correlation analysis between SFXN3 expression and key proteins in the Wnt/β-Catenin signaling pathway. (L) Subcellular fractionation followed by Western blotting was performed to assess β-catenin nuclear translocation. Cytoplasmic (Cyto) and nuclear (Nuc) fractions were probed for β-catenin, with β-actin (cytoplasmic marker) and Histon H3 (nuclear marker) used to confirm fractionation quality. Data are presented as mean ± SD. from three independent experiments ( n = 3). One-way ANOVA was used in (A, B, E), and two-way ANOVA was used in (F). *, p < 0.05.

Article Snippet: The ATCC supplied the AML cell lines THP-1, KG-1, U937, and K562, and the stromal cell line HS-5.

Techniques: Knockdown, Protein-Protein interactions, Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, DNA Synthesis, Activity Assay, Control, TUNEL Assay, Fractionation, Translocation Assay, Marker

Journal: Translational Oncology

Article Title: REST-driven upregulation of SFXN3 promotes AML progression via Wnt/β-catenin activation and confers decitabine resistance

doi: 10.1016/j.tranon.2026.102705

Figure Lengend Snippet: The Wnt/β-Catenin Pathway Agonist SKL2001 Reverses the Effects of SFXN3 Knockdown on Leukemia Cell Proliferation and Apoptosis. (A) Western blot analysis of SFXN3 protein expression following SFXN3 knockdown and treatment with SKL2001, to assess whether SKL2001 significantly modulates SFXN3 expression. (B) CCK-8 assays were performed to evaluate whether SKL2001 could reverse the inhibitory effects of SFXN3 knockdown on the proliferation of THP-1 and KG-1 leukemia cells. (C) EdU staining assays were used to assess DNA synthesis activity, analyzing the ability of SKL2001 to restore proliferation suppressed by SFXN3 knockdown, (bar=50 ųm). (D) Quantitative analysis of EdU fluorescence intensity to evaluate DNA replication across different treatment groups. (E) Western blot analysis of cell cycle regulators CDK4, CDK6, Cyclin D1, and Cyclin E1 to determine whether SKL2001 rescues the expression of these proteins in SFXN3-silenced cells. (F) Western blot analysis of pro-apoptotic proteins (BAX, BAK) and anti-apoptotic proteins (Bcl-2, Bcl-xl) to confirm that SKL2001 mitigates the apoptosis-promoting effects of SFXN3 knockdown. (G) TUNEL assays were conducted to assess whether SKL2001 suppresses the enhanced apoptosis induced by SFXN3 knockdown, (bar=50ųm). (H) Quantification of TUNEL fluorescence intensity, reflecting apoptosis levels under different treatment conditions. (I) Subcellular fractionation followed by Western blotting was performed to assess β-catenin nuclear translocation. Cytoplasmic (Cyto) and nuclear (Nuc) fractions were probed for β-catenin, with β-actin (cytoplasmic marker) and Histon H3 (nuclear marker) used to confirm fractionation quality. Data are presented as mean ± SD. from at least three independent experiments. One-way ANOVA was used in (D, H), and two-way ANOVA was used in (B). *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. control or scramble group.

Article Snippet: The ATCC supplied the AML cell lines THP-1, KG-1, U937, and K562, and the stromal cell line HS-5.

Techniques: Knockdown, Western Blot, Expressing, CCK-8 Assay, Staining, DNA Synthesis, Activity Assay, Fluorescence, TUNEL Assay, Fractionation, Translocation Assay, Marker, Control

Journal: Translational Oncology

Article Title: REST-driven upregulation of SFXN3 promotes AML progression via Wnt/β-catenin activation and confers decitabine resistance

doi: 10.1016/j.tranon.2026.102705

Figure Lengend Snippet: The REST–SFXN3 Axis Promotes Malignant Phenotypes in AML Cells via the Wnt/β-Catenin Signaling Pathway. (A) Western blot analysis of the effect of REST knockdown (sh-REST) on SFXN3 expression, and the reversal of this effect by SFXN3 overexpression. (B) CCK-8 assays assess the impact of sh-REST and SFXN3 overexpression on AML cell proliferation. (C) EdU incorporation assays evaluate the effects of sh-REST and SFXN3 overexpression on DNA synthesis activity in AML cells, (bar=50ųm). (D) Quantification of EdU-positive cells to compare DNA synthesis capacity across groups. (E) Western blot analysis of proliferation-related proteins CDK4, CDK6, Cyclin D1, and Cyclin E1 under sh-REST and SFXN3 overexpression conditions. (F) Band intensities were quantified using ImageJ software and normalized to the indicated internal controls. (G) TUNEL assays detect apoptotic cells after REST knockdown and SFXN3 overexpression, (bar=50ųm). (G) Quantitative analysis of apoptotic cells in THP-1 and KG-1 cell lines. (H) Quantification of TUNEL fluorescence intensity, reflecting apoptosis levels under different treatment conditions. (I) Western blot evaluation of pro-apoptotic proteins (BAX, BAK) and anti-apoptotic proteins (Bcl-2, Bcl-xl), demonstrating REST knockdown promotes apoptosis, which is reversed by SFXN3 overexpression. (J) Subcellular fractionation followed by Western blotting was performed to assess β-catenin nuclear translocation. Cytoplasmic (Cyto) and nuclear (Nuc) fractions were probed for β-catenin, with β-actin (cytoplasmic marker) and Histon H3 (nuclear marker) used to confirm fractionation quality. Data are presented as mean ± SD from three independent experiments ( n = 3).One-way ANOVA was used in (D, F,H), and two-way ANOVA was used in (B). **, p < 0.01; ***, p < 0.001.

Article Snippet: The ATCC supplied the AML cell lines THP-1, KG-1, U937, and K562, and the stromal cell line HS-5.

Techniques: Western Blot, Knockdown, Expressing, Over Expression, CCK-8 Assay, DNA Synthesis, Activity Assay, Software, TUNEL Assay, Fluorescence, Fractionation, Translocation Assay, Marker

Journal: Translational Oncology

Article Title: REST-driven upregulation of SFXN3 promotes AML progression via Wnt/β-catenin activation and confers decitabine resistance

doi: 10.1016/j.tranon.2026.102705

Figure Lengend Snippet: Decitabine Suppresses AML Cell Proliferation and Promotes Apoptosis via SFXN3 Inhibition. (A) RT-PCR analysis of the effects of Gefitinib, Disulfiram, and Decitabine on SFXN3 mRNA expression. (B) Western blot analysis of SFXN3 protein levels following treatment with Gefitinib, Disulfiram, and Decitabine. (C) CCK-8 assay to calculate the IC50 values of Decitabine in THP-1 and KG-1 cells, identifying appropriate drug concentrations for subsequent experiments (D) CCK-8 assays were performed to evaluate AML cell viability at 6,12,24,48, and 72 h following treatment with 50 nm decitabine, thereby determining the optimal treatment duration. E) EdU incorporation assay evaluating the proliferation capacity of AML cells after Decitabine treatment, (bar=50ųm). (F) Western blot analysis of proliferation-related proteins (P21, P27, CDK4, and CDK6) following Decitabine treatment. (G) TUNEL staining to detect DNA fragmentation at the 3′-OH ends, marking apoptotic cells after Decitabine exposure, (bar=50ųm). (H) Western blot analysis of pro-apoptotic (e.g., BAX, BAK) and anti-apoptotic (e.g., Bcl-2, Bcl-xl) protein expression in response to Decitabine. (I) Western blot analysis of key components of the Wnt/β-Catenin signaling pathway after Decitabine treatment, revealing pathway inhibition. Subcellular fractionation followed by Western blotting was performed to assess β-catenin nuclear translocation. Cytoplasmic (Cyto) and nuclear (Nuc) fractions were probed for β-catenin, with β-actin (cytoplasmic marker) and Histon H3 (nuclear marker) used to confirm fractionation quality. n = 3,Error bars indicate mean ± SD; One-way ANOVA in (D, F); **, p < 0.01, *** p <0.001.

Article Snippet: The ATCC supplied the AML cell lines THP-1, KG-1, U937, and K562, and the stromal cell line HS-5.

Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, CCK-8 Assay, TUNEL Assay, Staining, Fractionation, Translocation Assay, Marker

Journal: Scientific Reports

Article Title: The Caspase-1-EGR4 axis regulates macrophage repolarization in acute myeloid leukemia cells

doi: 10.1038/s41598-026-41381-x

Figure Lengend Snippet: Efficient CASP1 KD in THP-1 and MOLM-13 cells. ( A ) (left) GFP fluorescence and (right) FCM analysis of GFP expression levels in the stable polyclonal cell pools. ( B , C ) Evaluation of CASP1-KD efficiency by qPCR and western blot. Values represent mean ± SD of triplicate experiments. Statistical significance versus the shNC group is shown, with *, ***, and ns representing p < 0.05, p < 0.001, and not significant, respectively. Cropped blot images are shown. Groupings of lanes from non-adjacent parts of the same gel are indicated by black borders. Full-length, uncropped original blots are provided in Supplementary Dataset.

Article Snippet: The human AML cell lines THP-1 and MOLM-13 were purchased from Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Fluorescence, Expressing, Western Blot

Journal: Scientific Reports

Article Title: The Caspase-1-EGR4 axis regulates macrophage repolarization in acute myeloid leukemia cells

doi: 10.1038/s41598-026-41381-x

Figure Lengend Snippet: Flow cytometric analysis of macrophage polarization markers. The THP-1 ( A , B ) and MOLM-13 ( C , D ) models are presented in each panel with representative histograms (left) and quantitative data (right). ( A , C ) CD163 + M2 macrophages; ( B , D ) CD86 + M1 macrophages. Values shown are mean ± SD ( n ≥ 3 independent experiments). *, **, ***, or ns denotes p < 0.05, p < 0.01, p < 0.001,or not significant versus the shNC control, respectively.

Article Snippet: The human AML cell lines THP-1 and MOLM-13 were purchased from Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Control

Journal: Scientific Reports

Article Title: The Caspase-1-EGR4 axis regulates macrophage repolarization in acute myeloid leukemia cells

doi: 10.1038/s41598-026-41381-x

Figure Lengend Snippet: Transcriptomic profiling identifies EGR4 as a key transcriptional target upregulated by CASP1 KD in AML cells. ( A ) Heat plot of the global transcriptome from shNC and shCASP1 THP-1 cells. ( B ) Distribution of DEGs is shown in a volcano plot, where statistically significant upregulations and downregulations are highlighted in red and blue, respectively. ( C ) KEGG pathway (Kanehisa Laboratories, https://www.kegg.jp/ ) classification of the DEGs, showing the most enriched functional categories. ( D ) GO (The Gene Ontology Resource, http://geneontology.org/ ) enrichment analysis for biological processes, showing the most significantly enriched terms. ( E ) qPCR validation of EGR4 mRNA expression in THP-1 cells from the indicated groups. ( F , G ) EGR4 protein expression was assessed by western blot in THP-1 and MOLM-13 cells. We calculated the data from three independent experiments as the mean ± SD, using *** p < 0.001 as the threshold for statistical significance compared to the shNC group. Cropped blot images are shown. Groupings of lanes from non-adjacent parts of the same gel are indicated by black borders. Full-length, uncropped original blots are provided in Supplementary Dataset.

Article Snippet: The human AML cell lines THP-1 and MOLM-13 were purchased from Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Western Blot

Journal: Scientific Reports

Article Title: The Caspase-1-EGR4 axis regulates macrophage repolarization in acute myeloid leukemia cells

doi: 10.1038/s41598-026-41381-x

Figure Lengend Snippet: EGR4 KD rescues the macrophage polarization shift induced by CASP1 KD. ( A , C ) RT-qPCR analysis of EGR4 mRNA levels. ( B , D ) Representative immunoblot of EGR4 protein expression. ( E , F ) Representative immunoblots for CD206 and CD86 in macrophages following stimulation with CM from the indicated AML cell groups. ( G ) Representative immunofluorescence staining for CD206 (red) in macrophages.Nuclear staining was performed with DAPI (blue) (scale bar: 50 μm). ( H – K ) Flow cytometric analysis of ( H , J ) CD163 + and ( I , K ) CD86 + macrophage populations after treatment with CM from ( H , I ) THP-1 or ( J , K ) MOLM-13 models. Graphical summary shows representative histograms (left) and the corresponding quantification of positive cells (right), presented as mean ± SD ( n ≥ 3). Cropped blot images are shown. Groupings of lanes from non-adjacent parts of the same gel are indicated by black borders. Full-length, uncropped original blots are provided in Supplementary Dataset.

Article Snippet: The human AML cell lines THP-1 and MOLM-13 were purchased from Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: The Caspase-1-EGR4 axis regulates macrophage repolarization in acute myeloid leukemia cells

doi: 10.1038/s41598-026-41381-x

Figure Lengend Snippet: Mechanism of the CASP1-EGR4 axis through the IL-10/STAT3 pathway in macrophages. ( A , B ) Conditioned medium (CM) from THP-1 cells with CASP1 knockdown (KD) reduces the levels of IL-10 and phosphorylated STAT3 (p-STAT3), but not total STAT3, in recipient macrophages. ( A ) Representative western blots and ( B ) statistical summary. ( C , D ) CM from MOLM-13 cells with CASP1 KD reduces the levels of IL-10 and p-STAT3, but not total STAT3, in recipient macrophages. ( C ) Representative western blots and ( D ) statistical summary. ( E , F ) Additional KD of EGR4 in CASP1-KD THP-1 cells partially reverses the reduction of IL-10 and p-STAT3 in macrophages treated with the corresponding CM. ( E ) Representative western blots and ( F ) statistical summary. ( G , H ) Additional KD of EGR4 in CASP1-KD MOLM-13 cells partially reverses the reduction of IL-10 and p-STAT3 in macrophages treated with the corresponding CM. ( G ) Representative western blots and ( H ) statistical summary. Cropped blot images are shown. The STAT3 and p-STAT3 data are presented in separate frames to indicate they are from different blots. Full-length, uncropped original blots and, where informative, multiple exposures (e.g., for IL-10 and STAT3) are provided in Supplementary Dataset.

Article Snippet: The human AML cell lines THP-1 and MOLM-13 were purchased from Procell Life Science and Technology Co., Ltd (Wuhan, China).

Techniques: Knockdown, Western Blot