Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Chemical structure of UNC1666, with inhibition constant (K i ) of 0.16 nM for Mer (enzymatic IC 50 : 0.55 nM) and 0.67 nM for Flt3 (enzymatic IC 50 : 0.69 nM). (B) Chemical structure of UNC1653, which lacks significant activity against Mer (enzymatic IC 50 : 560 nM) and Flt3 (enzymatic IC 50 : 220 nM) and is used as a negative control in these studies. (C) Whole cell lysates from AML cell lines with known Flt3 mutation status were analyzed by immunoblot and demonstrate presence or absence of the Mer tyrosine kinase (above) and the Flt3 tyrosine kinase (middle). Actin is shown as an indicator of total protein (below).

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Inhibition, Activity Assay, Negative Control, Mutagenesis, Western Blot

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Mer was immunoprecipitated from AML cell lysates and phosphorylated ( p -Mer) and total Mer (~180 kDa) levels were assessed by immunoblot analysis. This representative blot of the Kasumi-1 cell line demonstrates decreased Mer phosphorylation after treatment with increasing doses of UNC1666. (B) Flt3 was immunoprecipitated from AML cell lysates and phosphorylated ( p -Flt3) and total Flt3 (130/160 kDa) levels were assessed by immunoblot analysis. This representative blot of the MV4;11 cell line demonstrates decreased Flt3 phosphorylation after treatment with increasing doses of UNC1666. (C) Inhibition of downstream signaling after administration of UNC1666 in a Mer expressing AML cell lines that does not express a Flt3-ITD mutation (Kasumi-1) compared with equivalent concentrations of vehicle (DMSO) or inactive control TKI UNC1653. Actin is shown as an indicator of total protein. (D) Downstream signaling after administration of UNC1666 in a Flt3-ITD AML cell line that does not express Mer (MV4;11). Representative blots from at least 3 independent experiments are shown. nM = nanomolar

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Inhibition, Expressing, Mutagenesis, Control

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: Mer pos or Flt3-ITD AML cell lines were treated with UNC1666, vehicle (DMSO), or inactive control TKI UNC1653 for 72 hours and then analyzed by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide to identify apoptotic and dead cells. (A) Representative flow cytometry profiles of Kasumi-1 cells are shown. The percentages of live (lower left quadrant), early apoptotic (lower right quadrant), and late apoptotic/dead cells (upper quadrants) are shown. (B) Graphic representation of flow cytometric analyses of apoptotic/dead cells. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, *** p < 0.001, NS = not significant. (C) Cells were treated as indicated for 72 hours, whole cell lysates were prepared and the indicated apoptotic proteins were assessed by immunoblot analysis. Actin is shown as a loading control.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Control, Flow Cytometry, Staining, Derivative Assay, Western Blot

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: Mer positive or Flt3-ITD AML cell lines were treated with UNC1666, vehicle (DMSO), or inactive control TKI UNC1653 for 72 hours, fixed with 100% ethanol and then analyzed by flow cytometry after staining with propidium iodide to identify stage of cell cycle. Graphic representation of cell cycle progress in Kasumi-1 and MV4;11 cells using ModFit analysis are shown. The percentages of cells in G2/M (light gray), S (white), and G1 (dark gray) phases are shown. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Control, Flow Cytometry, Staining, Derivative Assay

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Diagram of the replating assay. Cells were treated with UNC1666 or vehicle for 72 hours, then washed to remove any residual compound and equal numbers of viable cells (1.5 × 10 4 /ml) were replated in growth medium on Day 0. On Day 6 after replating, the number of viable cells was determined. (B) Graphic representation of the results of the replating assay in Mer or Flt3-ITD expressing cell lines demonstrating decreased rebound growth after treatment with UNC1666. Mean values and standard errors were derived from at least 3 independent experiments. (C) Colony-formation assays were performed using Mer or Flt3-ITD expressing AML cell lines. Cells were grown in soft agar with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle or negative control TKI UNC1653. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = not significant.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Expressing, Derivative Assay, Negative Control

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Immunoblot analysis of Mer and Flt3 expression in lysates prepared from AML patient samples. (B) Flt3 mutation status of patient samples determined by molecular profiling. (C, D) Dose-dependent inhibition of Mer and Flt3 phosphorylation in response to treatment with UNC1666. AML blasts from patient sample #10510 (Mer positive, Flt3-ITD high allelic ratio) were treated with UNC1666 or vehicle for two hours. (C) Mer and Flt3 were immunoprecipitated from cell lysates and phosphorylated Mer ( p -Mer), total Mer (~180 kDa), phosphorylated Flt3 ( p -Flt3) and total Flt3 (130/160 kDa) were detected by immunoblot. (D) Phosphorylation of downstream signaling molecules was assessed by immunoblot after treatment with UNC1666 or vehicle.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Phospho-proteomics, Immunoprecipitation

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Graphic representation of apoptosis and cell death in AML patient samples after treatment with UNC1666 or vehicle for 72 hours. Apoptotic and dead cells were determined by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide. Values derived from each sample are shown. (B) Colony-forming assays were performed in methylcellulose with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle. Mean values and standard errors derived from triplicate samples are shown. (C) Graphic representation of the effect of UNC1666 on normal cord blood colony forming potential. Mean values and standard errors were derived from 3 independent experiments. ** p < 0.01, NS = not significant.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Flow Cytometry, Staining, Derivative Assay

Journal: Redox Biology

Article Title: Macrophages and macrophage extracellular vesicles confer cancer ferroptosis resistance via PRDX6-mediated mitophagy inhibition

doi: 10.1016/j.redox.2025.103826

Figure Lengend Snippet: Macrophages upregulate glutathione metabolic pathways in cancer cells (A,B) Transcriptome analysis of the top 20 up-regulated KEGG enrichment pathways in MC38 sorted from the co-culture system without (A) or with (B) RSL3 treatment. (C – E) Transcription changes of glutathione metabolism genes in MC38 (C) , B16 (D) or CT26 (E) cells sorted from Mφ co-culture system. The statistical significance of the differences was assessed using the Student's t -test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Each point represents one of three independent experiments. Column bars show mean and SD. (F) Venn diagram of proteins numbers and overlap3ing proteins numbers from M1-EV and M2-EV by liquid chromatography-tandem mass spectrometry proteomic profiling. (G) Scatter diagram of overlaping proteins from M1-EV and M2-EV, and PRDX6 abundance. (H) PRDX6 expression in macrophages and Mφ-EV. The picture shows one of three representative experiments. Calnexin is presented as a positive marker for Mφ but a negative marker of EV. (I–N) Protein expression and quantification compared to β-actin of ACSL4, GPX4, xCT and PRDX6 in MC38 (I,L) , B16 (J,M) or CT26 (K,N) cells from the macrophage (I–K) or Mφ-EV (L – N) co-culture system with or without RSL3 treatment. We calculated the ratio of intensity of a given protein band to the intensity of beta-actin band, and treated the control lane (first lane in each immunoblotting) as intensity = 1. Data shown are from the representative experiment of three independent replicates.

Article Snippet: Cells were treated with RSL3 (Selleck, USA, #S8155), erastin (Selleck, USA, #S7242), FIN56 (MCE, USA, # HY103087 ), ML210 (MCE, USA, # HY100003 ), IKE (MCE, USA, # HY114481 ) or ferrostatin-1 (Selleck, USA, #S7243) to induce or inhibit ferroptosis, with P62-mediated mitophagy inducer (MCE, #HY-115576) or Mdivi (MCE, USA, #HY-15886) to induce or inhibit mitophagy, with MJ33 (MCE, USA, #HY-115062) to inhibit phospholipase A2 activity of PRDX6 and with MitoQ (MCE, USA, #HY-100116A) or MitoTempo (MCE, USA, #HY-112879) to deplete mitochondrial superoxide, with sodium selenite (MCE, USA, #HY-W686381) to provide selenium, with ML385 (MCE, USA, #HY-100523) to inhibit NRF2.

Techniques: Co-Culture Assay, Liquid Chromatography, Mass Spectrometry, Expressing, Marker, Control, Western Blot

Journal: Redox Biology

Article Title: Macrophages and macrophage extracellular vesicles confer cancer ferroptosis resistance via PRDX6-mediated mitophagy inhibition

doi: 10.1016/j.redox.2025.103826

Figure Lengend Snippet: Macrophages and Mφ-derived extracellular vesicles inhibit ferroptosis and promote PRDX6 expression in tumors (A) Timeline of clodronate liposome (CL) and RSL3 treatment for MC38 subcutaneous mouse model. (B – D) Harvested tumors (B), individual tumor growth curves (C) and average tumor growth curves (D) of MC38 tumors collected from mice treated with vehicle, CL, RSL3 or combination of both compounds, N = 5 per group. (E,F) Quantified percentage (E) and representative flow cytometry scatter diagrams (F) of F4/80 + macrophages in CD45 + immune cells gate from MC38 tumors. (G,H) Quantification of lipid peroxidation (G) and representative flow cytometry scatter diagrams of BODIPY/C11 dye (H) in MC38 tumors. (I) Representative immunohistology images of GPX4, 4-HNE, PRDX6 and F4/80 expression in the tumor samples. (J) Timeline of treatment of MC38 tumor-bearing mice with M2 macrophages extracellular vesicles (M2-EV) and RSL3. (K – M) Harvested tumor images (K), individual tumor growth curves (L) and tumor mean volume curves (M) of MC38 tumors collected from experiment shown in panel ( J ), N = 5 mice per group. (N,O) Quantified percentage (N) and representative flow cytometry scatter diagrams (O) of F4/80 + macrophages in CD45 + gate from MC38 tumors obtained from experiment ( J ). (P,Q) Quantified lipid peroxidation (P) and representative flow cytometry scatter diagrams of BODIPY/C11 dye (Q) of total cells from MC38 tumors. (R) Representative immunohistochemistry images of GPX4, 4-HNE, PRDX6 and F4/80 expression in the tumor samples from experiment depicted in panel ( J ). Data were analyzed with ANOVA and Tukey's comparing with each other group, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Column error bars show mean and SD.

Article Snippet: Cells were treated with RSL3 (Selleck, USA, #S8155), erastin (Selleck, USA, #S7242), FIN56 (MCE, USA, # HY103087 ), ML210 (MCE, USA, # HY100003 ), IKE (MCE, USA, # HY114481 ) or ferrostatin-1 (Selleck, USA, #S7243) to induce or inhibit ferroptosis, with P62-mediated mitophagy inducer (MCE, #HY-115576) or Mdivi (MCE, USA, #HY-15886) to induce or inhibit mitophagy, with MJ33 (MCE, USA, #HY-115062) to inhibit phospholipase A2 activity of PRDX6 and with MitoQ (MCE, USA, #HY-100116A) or MitoTempo (MCE, USA, #HY-112879) to deplete mitochondrial superoxide, with sodium selenite (MCE, USA, #HY-W686381) to provide selenium, with ML385 (MCE, USA, #HY-100523) to inhibit NRF2.

Techniques: Derivative Assay, Expressing, Flow Cytometry, Immunohistochemistry

Journal: Redox Biology

Article Title: Macrophages and macrophage extracellular vesicles confer cancer ferroptosis resistance via PRDX6-mediated mitophagy inhibition

doi: 10.1016/j.redox.2025.103826

Figure Lengend Snippet: PRDX6-dependent ferroptosis resistance relies on glutathione peroxidase activity (A) Scheme of the enzyme activities of PRDX6 and the strategies to inhibit them. (B – D) Flow cytometry analysis of RSL3-induced lipid peroxidation in B16 ( B ), CT26 ( C ) or MC38 ( D ) cells overexpressing (OE) PRDX6 versus negative control (NC) cells. The cells were treated with vehicle or 10 μM MJ33. (E) Diagram of PRDX6 overexpression plasmid and mutation site details of C47A, R132A and H39A mutant PRDX6. (F–K) Flow cytometry analysis of lipid peroxidation ( F–H ) and cell viability ( I–K ) in B16 ( F,I ), CT26 ( G,J ), MC38 ( H,K ) cells with PRDX6 knockdown. The cells were next transfected with wildtype (WT) or mutant PRDX6 (C47A, R132A, H39A) and treated with increasing concentrations of RSL3. Areas under the curve (AUC) were compared with one way ANOVA and Tukey's test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Column error bars show the mean and SD. (L – N) Analysis of GSH/GSSG in B16 ( L ), CT26 ( M ) or MC38 ( N ) cells with the PRDX6 manipulations following RSL3 treatment. Data were analyzed between the same treatment samples with ANOVA and Dunnett's test, using WT PRDX6 as control, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Column error bars show the mean and SD.

Article Snippet: Cells were treated with RSL3 (Selleck, USA, #S8155), erastin (Selleck, USA, #S7242), FIN56 (MCE, USA, # HY103087 ), ML210 (MCE, USA, # HY100003 ), IKE (MCE, USA, # HY114481 ) or ferrostatin-1 (Selleck, USA, #S7243) to induce or inhibit ferroptosis, with P62-mediated mitophagy inducer (MCE, #HY-115576) or Mdivi (MCE, USA, #HY-15886) to induce or inhibit mitophagy, with MJ33 (MCE, USA, #HY-115062) to inhibit phospholipase A2 activity of PRDX6 and with MitoQ (MCE, USA, #HY-100116A) or MitoTempo (MCE, USA, #HY-112879) to deplete mitochondrial superoxide, with sodium selenite (MCE, USA, #HY-W686381) to provide selenium, with ML385 (MCE, USA, #HY-100523) to inhibit NRF2.

Techniques: Activity Assay, Flow Cytometry, Negative Control, Over Expression, Plasmid Preparation, Mutagenesis, Knockdown, Transfection, Control

Journal: Redox Biology

Article Title: Macrophages and macrophage extracellular vesicles confer cancer ferroptosis resistance via PRDX6-mediated mitophagy inhibition

doi: 10.1016/j.redox.2025.103826

Figure Lengend Snippet: Cancer PRDX6 overexpression limits ferroptosis and reverses macrophage depletion effect (A-C) Tumor mean volume curves (A) , harvested tumor image (B) , and individual tumor growth curve (C) of MC38 tumors with PRDX6 overexpression (OE) or negative control (NC). The animals were treated with RSL3+PLX3397 (R + P) or vehicle. N = 5 per group. (D,E) Quantified percentage of F4/80 + macrophages in CD45 + gate (D) and representative flow cytometry scatter diagrams of F4/80 + macrophages (E) from MC38 tumors. (F,G) Quantified lipid peroxidation (F) and representative flow cytometry scatter diagrams of BODIPY/C11 dye (G) of total cells from MC38 tumors. (H) Representative immunohistochemistry images of PRDX6, 4-HNE and F4/80 expression in harvested tumor tissues. (I,J) Quantification of CD8 + and CD4 + T cells ratio (I) and representative flow cytometry scatter diagrams of CD8 + and CD4 + T cells (J) from MC38 tumors treatment. (K – M) Tumor means volume curve (K) , harvest tumor image (L) and individual tumor growth curve (M) of PRDX6 knockout (KO) and negative control (NC) MC38 tumors which are treated with single RSL3 (R) or RSL3+PLX3397 (R + P) or vehicle. n = 5 per group. (N,O) Quantified lipid peroxidation (N) and representative scatter diagrams of BODIPY/C11 dye (O) of MC38 tumors as indicated treatment. (P,Q) Quantified percentage (P) and representative flow cytometry scatter diagrams (Q) of F4/80 + macrophages in CD45 + immune cells from MC38 tumors as indicated. (R) Representative immunohistology images of 4-HNE, PRDX6 and F4/80 expression in the tumor slice of indicated six groups above. Data were analyzed with ANOVA and Tukey's comparing with each other group, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Column error bars show mean and SD.

Article Snippet: Cells were treated with RSL3 (Selleck, USA, #S8155), erastin (Selleck, USA, #S7242), FIN56 (MCE, USA, # HY103087 ), ML210 (MCE, USA, # HY100003 ), IKE (MCE, USA, # HY114481 ) or ferrostatin-1 (Selleck, USA, #S7243) to induce or inhibit ferroptosis, with P62-mediated mitophagy inducer (MCE, #HY-115576) or Mdivi (MCE, USA, #HY-15886) to induce or inhibit mitophagy, with MJ33 (MCE, USA, #HY-115062) to inhibit phospholipase A2 activity of PRDX6 and with MitoQ (MCE, USA, #HY-100116A) or MitoTempo (MCE, USA, #HY-112879) to deplete mitochondrial superoxide, with sodium selenite (MCE, USA, #HY-W686381) to provide selenium, with ML385 (MCE, USA, #HY-100523) to inhibit NRF2.

Techniques: Over Expression, Negative Control, Flow Cytometry, Immunohistochemistry, Expressing, Knock-Out

Journal: Redox Biology

Article Title: Macrophages and macrophage extracellular vesicles confer cancer ferroptosis resistance via PRDX6-mediated mitophagy inhibition

doi: 10.1016/j.redox.2025.103826

Figure Lengend Snippet: Macrophage extracellular vesicles and PRDX6 inhibited cancer cell mitophagy (A,B) Volcano plot (A) and quantification (B) of phosphorylated proteins in MC38 treated with M2-EV for 8h versus not treated MC38 cells. Data are merged from 3 independent experiments. (C) Top 10 up- and down-regulated KEGG pathways of M2-EV treated MC38 cells vs not treated controls. (D) Significantly down-regulated phosphorylated proteins in the mitophagy-animal term from panel ( C ). (E,F) Representative pictures of mitophagy, lysosome, and mitochondria fluorescence in M2-EV treated MC38 cells (E) or PRDX6 overexpressed (PRDX6 OE/OE) MC38 cells (F) treated with vehicle or RSL3. (G) SQSTM1, BNIP3, BNIP3L expression in PRDX6 KO or NC MC38 cells which treated with RSL3 or MitoTempo. Data shown are from the representative experiment of three independent replicates. (H) Representative morphology of mitochondria and mitophagy in negative control MC38 cells and PRDX6 knockdown (PRDX6 KD) MC38, PRDX6 OE MC38, M2-EV-treated or 100 μM MitoTempo-treated MC38 cells cultured with 2 μM RSL3. Data shown are from the representative replicate of three independent experiments.

Article Snippet: Cells were treated with RSL3 (Selleck, USA, #S8155), erastin (Selleck, USA, #S7242), FIN56 (MCE, USA, # HY103087 ), ML210 (MCE, USA, # HY100003 ), IKE (MCE, USA, # HY114481 ) or ferrostatin-1 (Selleck, USA, #S7243) to induce or inhibit ferroptosis, with P62-mediated mitophagy inducer (MCE, #HY-115576) or Mdivi (MCE, USA, #HY-15886) to induce or inhibit mitophagy, with MJ33 (MCE, USA, #HY-115062) to inhibit phospholipase A2 activity of PRDX6 and with MitoQ (MCE, USA, #HY-100116A) or MitoTempo (MCE, USA, #HY-112879) to deplete mitochondrial superoxide, with sodium selenite (MCE, USA, #HY-W686381) to provide selenium, with ML385 (MCE, USA, #HY-100523) to inhibit NRF2.

Techniques: Fluorescence, Expressing, Negative Control, Knockdown, Cell Culture

Journal: Journal of Hepatocellular Carcinoma

Article Title: Coilin Affects the Prognosis of Hepatocellular Carcinoma Through Cell Cycle and Apoptosis

doi: 10.2147/JHC.S500119

Figure Lengend Snippet: Expression of COIL in HCC cell lines and HCC tissue from hospital. The expression of COIL in liver cancer cell lines huh7, hepG2 and hep3B was higher than that in L02 by immunohistochemistry ( A ) and Western blotting ( B ). ( C ) The relative expression of COIL in HCC was significantly higher than that in normal liver tissue by qRT-PCR (***p value < 0.001). ( D ) The expression of COIL in HCC tissue was higher than that in normal liver tissue. ( E, F ) Representative immunohistochemistry results of low or high COIL expression. ( G ) High COIL expression was associated with poor overall survival.

Article Snippet: The human normal liver cell line L02 was obtained from Beijing Baiao Bowei Biotechnology Co., Ltd.

Techniques: Expressing, Immunohistochemistry, Western Blot, Quantitative RT-PCR