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: bioRxiv

Article Title: Programmable Lipid Nanoparticle Targeting via Corona Engineering

doi: 10.64898/2026.02.27.708523

Figure Lengend Snippet: ApoE mutants reduce ApoE-mediated LNP uptake and liver accumulation. (a) Schematic of dApoE precoating and haPCSK9 pretreatment strategies for modulating LNP biodistribution. (b) Representative fluorescence microscopy images showing uptake of GFP-loaded LNPs in HEK293FT cells after incubation with media containing wild-type (WT) ApoE or single, double, triple, and five-residue combinatorial ApoE mutants. Progressive combination of receptor-binding domain mutations resulted in increasingly strong inhibition of ApoE-mediated uptake, with the five-residue mutant exhibiting the most pronounced blockade. Scale bar, 100 µm. (c) Quantification of the LNP uptake by GFP plate-read measurements, confirming that while individual mutations partially reduced uptake, combinatorial mutations produced progressively stronger inhibition, with the five-residue ApoE mutant showing maximal suppression of LNP internalization. (d) Uptake of LNPs in HEK293FT and AML-12 (e) hepatocytes using purified proteins, comparing WT ApoE, three-residue ApoE mutant (dApoE-3m), five-residue ApoE mutant (dApoE-5m), and haPCSK9 pretreatment. Both dApoE and haPCSK9 robustly suppress ApoE-dependent uptake. (f) Proteomic analysis of LNP–protein corona composition showing that dApoE precoating stabilizes the corona and maintains mutant ApoE enrichment even after subsequent serum exposure, whereas LNPs incubated with 10% mouse serum alone recruit a broader range of serum proteins. (g) Serum competition assays demonstrating enhanced uptake with WT ApoE and persistent inhibition by dApoE at higher concentrations, consistent with LC-MS findings on corona stability. (h) In vivo bioluminescence images after 6h of LNP injection (0.3 mg/kg RNA) showing reduced hepatic accumulation with dApoE precoating (25× relative to LNP mRNA) or haPCSK9 pretreatment (40 µg per mouse, administered 15 min before LNP injection). (i) Quantification of bioluminescence across major organs (liver, spleen, kidneys, heart, lungs), showing selective reduction in liver uptake without redistribution to other tissues. All data in this figure are mean ± SEM (n=3).

Article Snippet: HEK293FT and AML-12 cells were purchased from ATCC.

Techniques: Fluorescence, Microscopy, Incubation, Residue, Binding Assay, Inhibition, Mutagenesis, Produced, Purification, Liquid Chromatography with Mass Spectroscopy, In Vivo, Injection

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: BRD4 inhibitor significantly promotes HCP5 super‐enhancer activity and expression. (A) Reverse transcription‐quantitative polymerase chain reaction analysis of HCP5 mRNA expression levels in acute myeloid leukemia cell lines (THP‐1 and HL‐60) treated with the BRD4 inhibitor GNE‐987. (B) Western Blot analysis showing BRD4 expression levels in different groups. (C) Representative agarose gel electrophoresis image and statistical quantification of ChIP‐qPCR products. Compared to the Ctrl group, * p < 0.05, ** p < 0.01. All cell experiments were repeated three times. BRD4, Bromodomain‐containing protein 4; HCP5, HLA Complex P5.

Article Snippet: The acute myeloid leukemia (AML) cell lines Tsuchiya Human Phagocyte‐1 (THP‐1) and Human Leukemia‐60 (HL‐60) (TIB‐202 and CCL‐240, ATCC), along with T‐ALL–specific cell lines Jurkat, CCRF‐CEM, MOLT‐4, and RPMI‐8402 (TIB‐152, CCL‐111, CRL‐1582, and CCL‐27, ATCC), were cultured in RPMI‐1640 medium (11875093, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (A5670701, Gibco) and maintained in an incubator at 37°C with 5% CO 2 .

Techniques: Activity Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis, ChIP-qPCR

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: Bromodomain‐containing protein 4 Inhibitor GNE‐987 Significantly Inhibits acute myeloid leukemia Cell Proliferation and Migration and Induces Apoptosis. (A) CCK‐8 assay measuring cell proliferation in each group at 0, 12, 24, 36, 48, 60, and 72 h, with absorbance detected at OD450. (B) Representative images of Live and Dead staining for each group, with a bar chart depicting the statistical analysis of cell death ratios. Bar = 50 μm. (C) Colony formation assay for each group, with a bar chart showing the statistical analysis of colony numbers. (D) Flow cytometry analysis of apoptosis levels in each group, with a bar chart depicting the statistical analysis of apoptosis rates. Compared with the Ctrl group, * p < 0.05, ** p < 0.01. All cell experiments were repeated three times.

Article Snippet: The acute myeloid leukemia (AML) cell lines Tsuchiya Human Phagocyte‐1 (THP‐1) and Human Leukemia‐60 (HL‐60) (TIB‐202 and CCL‐240, ATCC), along with T‐ALL–specific cell lines Jurkat, CCRF‐CEM, MOLT‐4, and RPMI‐8402 (TIB‐152, CCL‐111, CRL‐1582, and CCL‐27, ATCC), were cultured in RPMI‐1640 medium (11875093, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (A5670701, Gibco) and maintained in an incubator at 37°C with 5% CO 2 .

Techniques: Migration, CCK-8 Assay, Staining, Colony Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: Silencing of HLA Complex P5 Reverses the Inhibitory Effect of GNE‐987 on acute myeloid leukemia Cell Viability. (A) Reverse transcription‐quantitative polymerase chain reaction results verify the silencing efficiency of sh‐HCP5. (B) CCK8 assay measuring cell proliferation at 0, 12, 24, 36, 48, 60, and 72 h, with absorbance detected at OD450. (C) Representative images of Live and Dead staining in each group and a bar chart showing the death ratio; scale bar = 50 μm. (D) Colony formation assay and statistical graph of colony numbers for each group. (E) Flow cytometry analysis of apoptosis levels and a statistical graph of apoptosis rates in each group. For panel A, compared with the sh‐NC group, * p < 0.05, ** p < 0.01. For panels B‐E, compared with the Ctrl group, * p < 0.05, ** p < 0.01; compared with the GNE‐987+sh‐NC group, # p < 0.05, ## p < 0.01. All cell experiments were repeated three times.

Article Snippet: The acute myeloid leukemia (AML) cell lines Tsuchiya Human Phagocyte‐1 (THP‐1) and Human Leukemia‐60 (HL‐60) (TIB‐202 and CCL‐240, ATCC), along with T‐ALL–specific cell lines Jurkat, CCRF‐CEM, MOLT‐4, and RPMI‐8402 (TIB‐152, CCL‐111, CRL‐1582, and CCL‐27, ATCC), were cultured in RPMI‐1640 medium (11875093, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (A5670701, Gibco) and maintained in an incubator at 37°C with 5% CO 2 .

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, CCK-8 Assay, Staining, Colony Assay, Flow Cytometry

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (A) Diagram illustrating how CD14 + monocytes were differentiated to MDMs and polarized. CD14 + monocytes were isolated from healthy blood donors, as a model for M2-like Mφs. AML cells were treated with DNR either in monoculture (-Mφ), in the presence of 50% macrophage conditioned media (Mφ-CM) or in direct co-culture with macrophages (Mφ). Representative plots of flow cytometric analysis of Annexin V-FITC and Viability Dye eFluor™ 450 fluorescence are shown for each cell line (Bi, Ci and Di). The treatment conditions are as follows; (Bii) U937: 0.25μM DNR for 24 hours (n=3), (Cii) THP-1: 0.125μM DNR for 72 hours (n=3), (Dii) KG-1a: 3μM DNR for 48 hours (n=4). AML survival (% of non-treated [NT]) was determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-FITC. Cells were analysed using the MACSQuant® Analyzer 10. Data are Mean ± SEM and were analysed using a one-way ANOVA followed by Tukey’s multiple-comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Isolation, Co-Culture Assay, Fluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (A) Mφs were loaded with MTDR on Day 9 of culture of M2-like Mφs. CFSE-labelled U937, KG1a and THP-1 were then co-cultured with Mφs at a 1:1 ratio with M2-like Mφs for 24, 48 and 72 hours respectively. Mitochondrial transfer was then assessed in CFSE-labelled FVD - viable AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10. (Bi) Representative histograms of flow cytometric analysis of MTDR fluorescence, U937 = unlabeled U937 cells; U937 + Mφ = MTDR signal from U937 cells in co-culture with M2-like Mφs; U937 + Mφ = MTDR signal from M2-like Mφs in co-culture with U937 cells and Mφ = MTDR signal from M2-like Mφs. (Bii) Data are Mean ± SEM of n=5 and analysed by a Mann-Whitney U Test, **p<0.01. (Ci) Representative histograms of flow cytometric analysis of MTDR fluorescence. (Cii) AML cell lines were cultured on and off MTDR loaded Mφs in the presence or absence of DMSO (vehicle control) or DNR or Ara-C or the combination of DNR & Ara-C, for the indicated times and concentrations: U937: 0.25μM DNR for 24 hours; THP-1: 0.125μM DNR for 72 hours; KG-1a: 3μM DNR for 48 hours, the same times for Ara-C at 2.5μM for all AML cell lines (n=3-4). Data were then analyzed by a one-way ANOVA followed by Dunnett’s multiple-comparison test, *p<0.05, **p<0.01. (D) Primary AML cells were cultured on and off MTDR loaded Mφs in the presence or absence of 0.125μM of DNR for 24h and mitochondrial transfer assessed as above (n=4). (E) U937 cells can interact with macrophages via TNTs. Blue (top left) is indicative of DNA staining (DAPI), green (top right) is the actin staining (ActinGreen488), red (bottom left) corresponds to mitochondria (MTDR), and the merged image is shown bottom right (n=1). Scale bar = 10 μm. White arrows indicate TNTs. (F) U937 cells were cultured alone or cultured either indirectly (tMφ, via transwell inserts) or directly with MTDR loaded Mφs for 24h. (G) U937 cells were cultured on MTDR loaded Mφs in the presence or absence of DNR or 1 μM Cyto B or the combination of DNR & Cyto B, for 24 hours. Mitochondrial transfer was then assessed in CFSE-labelled AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10 (n=6), and analysed by a one-way ANOVA followed by Tukey’s multiple-comparison test, *p<0.05, ns = non-significant. (Hi) Representative plots of flow cytometric analysis of Annexin V-FITC and Viability Dye eFluor™ 450 fluorescence. (Hii) U937 cells were treated with DNR +/- cyto B either in monoculture (-Mφ) or in direct co-culture with macrophages (+Mφ) for 24h. AML survival (% of NT) was determined by determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-APC. Cells were analysed using the MACSQuant® Analyzer 10 (AML cell lines). (H) Data are Mean ± SEM of n=4 and analysed by a two-way ANOVA followed by followed by Tukey’s multiple comparisons test, **p<0.01, ***p<0.0001, ns = non-significant, ns = non-significant.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Cell Culture, Fluorescence, Co-Culture Assay, MANN-WHITNEY, Control, Comparison, Staining

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (Ai) U937 cells were grown with and without M2-like monocyte-derived Mφs for 24 hours and then analyzed independently, using the Seahorse XFp Analyzer with the Mito Stress Test Kit. Sequential injections of Oligomycin (O), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (F), and Rotenone (R) were used to obtain respiration dynamics presented in panel (Aii). Data are mean ± SEM of n=3 and were then analyzed by using a paired student’s t-test, **p<0.01. (Bi) Representative histograms of flow cytometric analysis of CellROX fluorescence. (Bii) CFSE-labelled U937 were co-cultured at a 1:1 ratio with M2-like Mφs and in the presence of absence of DNR for 24 hours. Total ROS levels were then assessed in CFSE-labelled U937 cells using CellROX, which was analysed in the R2 (APC-Cy7) channel, via the MACSQuant® Analyzer 10. Relative CellROX values were calculated as a percentage of values obtained for U937 cells cultured alone in cRPMI (media con). The data are Mean ± SEM of n=3 and analysed by a two-way ANOVA followed by Tukey’s multiple-comparison test, **p<0.01, **p<0.001.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Derivative Assay, Fluorescence, Cell Culture, Comparison

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (A) CFSE-labelled U937 cells were directly cultured either in the presence (co-culture) or absence (monoculture) of M2-like Mφs for 24h. CD14 - U937 AML cells were then isolated from CD14 + Mφs, via the use of CD14 magnetic activated cell sorting. Proteomic profiling was then conducted on purified AML whole cell lysates (WCLs), via a TMT11-plex. Data shows the average values of n=4. (B) Association of transcript expression levels of RhoC and (C) Cofilin-1 with overall survival of patients with AML, plots were generated from interrogation of TCGA data via the GEPIA database. (Di) U937 were exposed to 0.25 mM DNR for up to 24h. Levels of ser3 p-cofilin and total cofilin (t-cofilin) were then assessed in purified U937 WCLs by immunoblotting. (Dii) Fold change of normalised ser3 p-cofilin to t-cofilin densitometry values are shown. The data are Mean ± SEM of n=5 and analysed by a one-way ANOVA followed by Dunnett’s multiple comparisons test, *p<0.05. (E) TCGA data were analyzed using GEPIA, and the expression of LIMK2 in AML compared to the normal samples is shown. (Fi) U937 were exposed to 0.25 mM DNR in the presence or absence of DMSO or 10mM of TH-257 for 12h. Levels of ser3 p-cofilin and total cofilin (t-cofilin) were then assessed in purified U937 WCLs by immunoblotting. (Fii) Fold change of normalised ser3 p-cofilin to t-cofilin densitometry values are shown. The data are mean ± SEM of n=3 and analysed by a one-way ANOVA followed by a Tukey’s multiple comparisons test, *p<0.05, **p<0.01. (G) CFSE-labelled U937 cells were cultured in the presence or absence of DNR, DMSO or 10 mM TH-257 either off (-Mφ) or on (+Mφ) MTDR loaded Mφs in the presence or absence of DNR for 24 hours. Mitochondrial transfer was then assessed in CFSE-labelled AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10 (n=3), and analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test, *p<0.05, ns = non-significant. (H) AML survival (% of NT) was determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-APC. Data are Mean ± SEM of n=4 and were then analyzed using a two-way ANOVA followed by Tukey’s multiple comparisons test, *p<0.05, ***p<0.001, ns = non-significant. (I) CFSE-labelled U937 cells were cultured in the presence or absence of DNR, water or 5 mM metformin either off (-Mφ) or on (+Mφ) MTDR loaded Mφs in the presence or absence of DNR for 24 hours. Mitochondrial transfer was then assessed in CFSE-labelled AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10 (n=5) with the data analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test, *p<0.05, **p<0.01, ns = non-significant. (J) AML survival (% of NT) was determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-APC. Data are Mean ± SEM of n=4 and were then analyzed using a two-way ANOVA followed by Tukey’s multiple-comparison test, **p<0.01, p<0.0001, ns = non-significant.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Cell Culture, Co-Culture Assay, Isolation, FACS, Purification, Expressing, Generated, Western Blot, Staining, Comparison