Journal: medRxiv

Article Title: The miR-362-3p/ BCLAF1 axis regulates cisplatin sensitivity and metastatic progression in triple-negative breast cancer

doi: 10.64898/2026.03.09.26347941

Figure Lengend Snippet: A. Negative correlation between miR-362-3p and BCLAF1 expression in TCGA breast cancer cohort. B-D. qRT-PCR analysis of BCLAF1 and SLC44A1 mRNA levels following miR-362-3p modulation in vitro . MiR-362-3p levels inversely suppressed BCLAF1 mRNA, while SLC44A1 remains unaffected. E. Western blotting demonstrating reciprocal changes in BCLAF1 protein expression, but not SLC44A1, upon miR-362-3p overexpression (OE) or inhibition. F-G. Validation of miR-362-3p-mediated regulation of BCLAF1 mRNA ( F ) and protein ( G ) in endpoint xenograft tumors. H The densitometric quantification of the 145 kDa BCLAF1 isoform (upper band). Data represent mean±SD. * and ** represent p <0.05 and p <0.01, respectively. Non-targeting controls, NTC; empty vector, EV.

Article Snippet: Membranes were incubated with the following primary antibodies: GAPDH (14C10) rabbit monoclonal antibody (Cell Signaling Technology, #2118, RRID:AB_561053), BCLAF1 polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, #26809-1-AP, RRID:AB_2880642), phospho-Histone H2A.X (Ser139) (20E3) rabbit monoclonal antibody (Cell Signaling Technology, #9718, RRID:AB_2118009), and SLC44A1 polyclonal antibody (Proteintech Group, Inc, #33135-1-AP, RRID: AB_10696180).

Techniques: Expressing, Quantitative RT-PCR, In Vitro, Western Blot, Over Expression, Inhibition, Biomarker Discovery, Plasmid Preparation

Journal: medRxiv

Article Title: The miR-362-3p/ BCLAF1 axis regulates cisplatin sensitivity and metastatic progression in triple-negative breast cancer

doi: 10.64898/2026.03.09.26347941

Figure Lengend Snippet: A. Western blotting demonstrating that miR-362-3p overexpression (OE) suppresses BCLAF1 and further potentiates cisplatin-induced γ-H2AX accumulation in vitro . B. Western blot showing that miR-362-3p inhibition in MDA-MB-436 cells attenuates γ-H2AX expression following cisplatin treatment. C. Functional rescue of cisplatin sensitivity in miR-362-3p-inhibited cells following BCLAF1 knockdown using two independent shRNAs. Data represent mean±SD. # , ## and ### represent adj p <0.05, adj p <0.01, and adj p <0.001, respectively. Non-targeting controls, NTC; empty vector, EV.

Article Snippet: Membranes were incubated with the following primary antibodies: GAPDH (14C10) rabbit monoclonal antibody (Cell Signaling Technology, #2118, RRID:AB_561053), BCLAF1 polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, #26809-1-AP, RRID:AB_2880642), phospho-Histone H2A.X (Ser139) (20E3) rabbit monoclonal antibody (Cell Signaling Technology, #9718, RRID:AB_2118009), and SLC44A1 polyclonal antibody (Proteintech Group, Inc, #33135-1-AP, RRID: AB_10696180).

Techniques: Western Blot, Over Expression, In Vitro, Inhibition, Expressing, Functional Assay, Knockdown, Plasmid Preparation

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: AC021683.2 knockdown accelerates ubiquitination and degradation of BCLAF1 in HL60-ADR cells under treatment with Ara-C (A) RT-qPCR was used to detect the subcellular fractions of AC021683.2 in HL60-ADR cells. U6 and β-actin were used as markers of the nucleus and cytoplasm, respectively. (B) Silver staining of AC021683.2 RNA pull-down proteins. A probe non-specific for AC021683.2 was used as control. Mass spectrometry analysis identified BCLAF1 (red arrow) as a protein that interacts with AC021683.2 . (C) AC021683.2 RNA pull-down followed by western blot validated the interaction with BCLAF1. (D) RT-qPCR analysis of BCLAF1 mRNA levels in control and AC021683.2 knockdown HL60-ADR cells in the presence of 5 μM Ara-C for 48 h (Data are represented as mean ± S.D. from triplicate experiments). (E) Western blotting analysis of BCLAF1 protein levels in control or AC021683.2 -knockdown HL60-ADR cells treated with or without (5 μM) Ara-C for 48 h. GAPDH was used as the internal control. (F) Western blotting analysis of BCLAF1 expression levels in AC021683.2 knockdown or control HL60-ADR cells treated with or without MG132 (20 μM) for 8 h in the presence of 5 μM Ara-C. GAPDH was used as the internal control. (G) The ubiquitination of BCLAF1 was analyzed by immunoprecipitation in AC021683.2 -knockdown Flag-BCLAF1 HL60 cells treated with or without (5 μM) Ara-C and western blot with indicated antibodies. Cell lysates were subjected to immunoblot with FLAG tag antibody. (H) AC021683.2 depleted or control HL60-ADR cells were treated with or without (5 μM) Ara-C for 48 h, and immunoprecipitations were performed with indicated antibodies. ns, no significance.

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Knockdown, Ubiquitin Proteomics, Quantitative RT-PCR, Silver Staining, Control, Mass Spectrometry, Western Blot, Expressing, Immunoprecipitation, FLAG-tag

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: BCLAF1 suppresses chemosensitivity of HL60-ADR cells to Ara-C (A) BCLAF1 expression in AML and normal group from GEPIA database. (B) Association between survival of AML patients and BCLAF1 expression levels from the TCGA database. p values were assessed using Gehan-Breslow-Wilcoxon test. (C) RT-qPCR analysis of mRNA levels of BCLAF1 in control and BCLAF1- overexpressing HL60 cells. (D) Western blotting analysis of BCLAF1 protein levels in control and BCLAF1-overexpressing HL60 cells. (E) RT-qPCR analysis of mRNA levels of BCLAF1 in control and BCLAF1 inhibited HL60-ADR cells. (F) Western blotting analysis of BCLAF1 protein levels in control and BCLAF1 knockdown HL60-ADR cells. (G) BCLAF1 overexpressed HL60 cells or control cells were treated with different concentrations of Ara-C for 48 h, the cell viability was detected by CCK-8 assay and the IC50 was calculated. (H) BCLAF1 depleted HL60-ADR cells or control cells were treated with different concentrations of Ara-C for 48 h, the cell viability was detected by CCK-8 assay and the IC50 was calculated. (I) BCLAF1 overexpressed HL60 cells or control cells were treated with or without Ara-C (5 μM) for 48 h, the cell apoptosis was detected by flow cytometry. (J) BCLAF1 knockdown HL60-ADR cells or control cells were treated with or without (5 μM) Ara-C for 48 h, the cell apoptosis was measured by flow cytometry. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-tailed Student’s t tests (C, E) and two-way ANOVA (G-J).

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Knockdown, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: AC021683.2 depletion enhances the chemosensitivity of HL60-ADR cells to Ara-C partially by depending on BCLAF1 (A) The cell viability was detected by CCK8 assay in AC021683.2 knockdown HL60-ADR cells with BCLAF1 overexpression stimulated with different concentrations of Ara-C for 48 h. (B) The cell viability was detected by CCK8 assay in BCLAF1 overexpressed HL60 cells with AC021683.2 knockdown stimulated with different concentrations of Ara-C for 48 h. (C) The indicated cell lines (control, AC021683.2 shRNA, AC021683.2 shRNA with BCLAF1 overexpression) were stimulated with (5 μM) Ara-C for 48 h, the cell apoptosis was measured by flow cytometry. (D) The indicated cell lines (control, BCLAF1 overexpression, BCLAF1 overexpression with AC021683.2 shRNA) were treated with (5 μM) Ara-C for 48 h, the cell apoptosis was measured by flow cytometry. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-way ANOVA (A–D).

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: CCK-8 Assay, Knockdown, Over Expression, Control, shRNA, Flow Cytometry

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: AC021683.2 or BCLAF1 is positively correlated with RAD50 in AML (A) The KEGG pathway enrichment analysis for the targets of the identified AC021683.2 from ENCORI database. (B) RT-qPCR analysis of mRNA levels of BRCA1 , ATM , RAD51 , RAD54 , and RAD50 in control and BCLAF1 suppressed HL60-ADR cells in the presence of 5 μM Ara-C. (C) RT-qPCR analysis of mRNA levels of BRCA1 , ATM , RAD51 , RAD54 , and RAD50 in control and AC021683.2 inhibited HL60-ADR cells in the presence of 5 μM Ara-C. (D) Co-expression analysis of RAD50 and BCLAF1 was performed in AML samples based on the ENCORI database. (E) Co-expression analysis of RAD50 and BCLAF1 was performed in AML samples based on the GEPIA database. (F) Co-expression analysis of AC021683.2 and RAD50 in AML samples was performed based on the ENCORI database. (G) Co-expression analysis of AC021683.2 and RAD50 in AML samples was performed based on the GEPIA database. (H) Pearson correlations between AC021683.2 and RAD50 expression levels in AML patient samples. (I) Western blotting analysis of BCLAF1 and RAD50 protein levels in control and BCLAF1 knockdown HL60-ADR cells in the presence of 5 μM Ara-C, respectively. (J) Western blotting analysis of BCLAF1 and RAD50 protein levels in AC02683.2 suppressed HL60-ADR cells and control cells under stimulation with 5 μM Ara-C. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-way ANOVA (B–C). ns, no significance.

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Quantitative RT-PCR, Control, Expressing, Western Blot, Knockdown

Journal: iScience

Article Title: LncRNA AC021683.2 promotes chemotherapy resistance in acute myeloid leukemia

doi: 10.1016/j.isci.2025.113439

Figure Lengend Snippet: RAD50 mediates the role of AC021683.2 and BCLAF1 in Ara-C-induced HL60-ADR cells apoptosis (A) RT-qPCR analysis of mRNA levels of RAD50 in control and RAD50 suppressed HL60-ADR cells. (B) Western blotting analysis of RAD50 protein levels in HL60-ADR cells with RAD50 knockdown and control cells. (C) RAD50 depleted HL60-ADR cells or control cells were treated with different concentrations of Ara-C for 48 h, respectively, the cell viability was detected by CCK-8 assay and the IC50 was calculated. (D) RAD50 knockdown HL60-ADR cells or control cells were treated with (5 μM) Ara-C for 48 h, respectively, the cell apoptosis was measured by Flow cytometry. (E) Western blotting analysis of RAD50 protein levels and BCLAF1-Flag in BCLAF1 overexpressed HL60 cells with RAD50 knockdown and control cells. (F) The cell viability was detected by CCK8 assay in BCLAF1 overexpressed HL60 cells with RAD50 knockdown stimulated with different concentrations of Ara-C for 48 h. (G) The indicated cell lines (Control, BCLAF1 overexpression, BCLAF1 overexpression with RAD50 shRNA) were treated with (5 μM) Ara-C for 48 h, respectively, the cell apoptosis was measured by flow cytometry. (H) Western blotting analysis of RAD50 protein levels in AC021683.2 knocked down HL60-ADR cells with RAD50 overexpression and control cells. (I) The cell viability was detected by CCK8 assay in AC021683.2 knocked down HL60-ADR cells with RAD50 overexpression stimulated with different concentrations of Ara-C for 48 h. (J) The indicated cell lines (Control, AC021683.2 shRNA, AC021683.2 shRNA with RAD50 overexpression) were stimulated with (5 μM) Ara-C for 48 h, respectively, the cell apoptosis was measured by Flow cytometry. Data are represented as mean ± S.D. from triplicate experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. p values were assessed using two-tailed Student’s t tests (A) and two-way ANOVA (C-D, F-G, and I-J). ns, no significance.

Article Snippet: BCLAF1 (Cat# 26809-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Hsp90AA1(Cat# 13171-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), RAD50(Cat# 29390-1-AP; Proteintech Group, Inc., Rosenont, IL, USA), Flag-tag (Cat# F3165; Sigma-Aldrich, Darmstadt, Germany), Ub (Cat# A19686; ABclonal, Wuhan, China), GAPDH (Cat# 60004; Proteintech Group, Inc. , Rosenont, IL, USA), Goat anti-Mouse IgG (H+L) HRP Secondary Antibody (Cat# 31430; Invitrogen, Carlsbad, CA, USA), Goat anti-Rabbit IgG (H+L) HRP Secondary Antibody (Cat# 31460; Invitrogen, Carlsbad, CA, USA), Ki-67 Antibody (Cat# GB111499 ; Servicebio, Wuhan, China), DAPI (Cat# G1012; Servicebio, Wuhan, China), TUNEL Assay Kit(Cat# G1504; Servicebio, Wuhan, China).

Techniques: Quantitative RT-PCR, Control, Western Blot, Knockdown, CCK-8 Assay, Flow Cytometry, Over Expression, shRNA, Two Tailed Test