human aml cell lines k562  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: Treatment with daunorubicin increases autophagy in K562 cells. ( A ) Schematic representation of the autophagy process with its components and related molecules. ( B ) K562 cells were treated with different indicated doses of daunorubicin (DNR) or vehicle (DMSO) for 24 h. ( C , D ) K562 cells were treated with 2 µM of daunorubicin for 24 h and 10 µM of chloroquine (CQ) for the last 4 h. ( B , C ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated conditions, and in grey: CQ conditions compared with DNR and CQ conditions). ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown.

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Western Blot, Expressing, Control, Software, Labeling, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: Treatment with 3-MA partially prevents the effects of DNR on autophagy and cell growth. K562 were treated with 2 µM of daunorubicin and 3 mM of 3-methyladenine (3-MA). ( A ) Proteins were extracted after 24 h, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, in black: all the conditions compared with the untreated conditions, and in grey: DNR conditions compared with DNR and 3-MA conditions). ( B ) Viable cells were counted in the presence of Trypan Blue after 1, 2, 3, and 4 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (***, p < 0.001).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Western Blot, Expressing, Control, Software

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p inhibits autophagy induced by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h and with 10 µM of chloroquine (CQ) for the last 16 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR. Normalization was completed with the endogenous control RNU44 . ( B ) Proteins were extracted, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( A , C , D ) The untreated scrambled condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p decreases the downregulation of cell growth by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (miR-15a) or scrambled mimic (Sc) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h ( A ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (*, p < 0.05, ***, p < 0.001). ( B ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. The untreated scrambled condition was set to 1. The normalized means are shown with SEM (**, p < 0.01).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Transfection, Control, Viability Assay, Luminescence Assay

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: Inhibition of mir-15a-5p induces autophagy. ( A – C ) K562 cells were transiently transfected with miR-15a-5p inhibitor (15ai) or scrambled inhibitor (Sci) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR and normalized with the expression of RNU44 . ( B ) Protein were extracted, and level of autophagy was analyzed by Western blot with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3B (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( D , E ) K562 cells were transiently transfected with miR-15a-5p inhibitor (miR-15ai) or scrambled inhibitor (Sci) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h. ( D ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. ( E ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. ( A – C , E ) The untreated scrambled conditions were set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, all the conditions compared with the untreated scrambled condition). ( D ) The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (**, p < 0.01).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Inhibition, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining, Viability Assay, Luminescence Assay

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p directly regulates the expression of autophagy target genes. ( A , B ) K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control and treated with 2 µM of daunorubicin (DNR) for 24 h before protein and RNA extraction. ( A ) Protein expression of ATG9A, ATG14, and GABARAPL1 was analyzed by Western blotting. Expression of β-actin was also detected as a loading control. ( B ) The expression of ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of a housekeeping gene RPLP0. ( C ) HEK 293T cells were co-transfected with wild-type (WT) or mutated (Mut∆) pMIR-luciferase vector, with either miR-15a-5p mimic or the scrambled oligonucleotide as control, and the pEF-β-galactosidase vector as internal control. After 24 h of transfection, cells were lysed, and the luminescence and β-gal activities were measured. ( B , C ) The normalized means of three independent experiments are shown with SEM. The scrambled condition was set to 1. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions, n.s.: non-significant).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Expressing, Transfection, Control, RNA Extraction, Western Blot, Quantitative RT-PCR, Luciferase, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p regulates autophagy by targeting autophagy genes. K562 cells were transiently co-transfected with miR-15a-5p inhibitor or scrambled inhibitor as control and with either a combination of four siRNA-targets ( ATG9A, ATG14, GABARAPL1, and SMPD1 ) or a siRNA-control for 24 h before protein and RNA extractions. ( A ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Protein expression of ATG9A, ATG14, and GABARAPL1 was also analyzed, as well as the expression of β-actin as a loading control. ( B ) In all the conditions, the expression of miR-15a-5p, ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of RNU44 for miR-15a-5p or with RPLP0 for the autophagy target genes. The scrambled inhibitor and siRNA condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, all the conditions compared with the scrambled condition).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

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