Figure 1 | Blood Cancer Journal

Figure 1

From: Macrolide antibiotics exert antileukemic effects by modulating the autophagic flux through inhibition of hERG1 potassium channels

Figure 1

Effects of MAs Clarithromycin (Cla), Erythromycin (Er) and hERG1 blockade and silencing on autophagy in acute leukemia cells. (a) LD50 values of Cla and Er in a panel of myeloid and lymphoid (AML cells: FLG 29.1, HL60; B cell precursor (BCP)-ALL cells: 697, REH) cell lines. Cells were treated with different concentrations of Cla and Er for 48 h and analyzed through the Annexin V/PI test. LD50 values were evaluated by nonlinear regression analysis using Origin 6 software (Microcal Software). Values are mean±s.e.m. of three indipendent experiments each performed in triplicate. (b) Left panel. Western blot of light chain enhancer 3 (LC3) expression (18 and 16 kDa bands) in FLG 29.1 cells treated with 56 μm Cla (the LD50 value as shown in a and in Supplementary Table 1S) or for different time points. LC3 was determined, as a biomarker of the autophagic process. Reprobing of the membrane was with an anti-tubulin antibody. Densitometric analysis is reported in Supplementary Figure S2. Right panel. Western blot of LC3 expression (18 and 16 kDa bands) in 697 cells treated with 78 μm Er (the LD50 value as shown in Figure 2) for 2 h. Membrane reprobing as above. Densitometric analysis is reported in Supplementary Figure S2. (c) Cyto-ID flow-cytometry analysis (representative panels) of FLG 29.1 and 697 cells treated with Cla (upper panel) and Er (lower panel) at their LD50 value for 2 h. The method is detailed in Materials and Methods. The mean autophagy activity factor (AAF) value, calculated in three separate experiments from AAF= 100 × [(MFI-treated cells−MFI-untreated cells)/MFI-treated cells] is shown on the top of each panel. (d) Western blot of pERK1/2 (top) and pAkt (bottom) in FLG 29.1 cells treated for 30 min with 56 μm Cla. Membrane reprobing (with anti-ERK1/2 and total Akt antibodies) as in b. Densitometric analysis is reported in Supplementary Figure S2. (e) Western blot of caspase 3 in FLG 29.1 treated for 30 min with 56 μm Cla. Membrane reprobing as in b. Densitometric analysis is reported in Supplementary Figure S2. (f) Left panel. Western blot of LC3 expression in FLG 29.1 cells treated with 22 μm E4031 for different time points: hERG1 blockade by E4031 induced increased levels of LC3II at least 2 h after treatment and last at least up to 24 h. Membrane reprobing was performed as in b. Densitometric analysis is reported in Supplementary Figure S2. Right panel. Western blot of LC3 expression in FLG 29.1 cells treated with CD160130, at the LD50 value (3.5 μm) for 2 h. Membrane reprobing and was performed as in b. (g) Cyto-ID flow-cytometry analysis (representative panels) of FLG 29.1 in control conditions and after 2 h treatment with 22 μm E4031. The AAF value, calculated as in c is reported on the top. Data shown are representative of two independent experiments. (h) Western blot of LC3 expression in FLG 29.1-sh7 and FLG 29.1-plKo. Membrane reprobing was performed as in b. Densitometric analysis is reported in Supplementary Figure S2. (i) Cyto-ID flow-cytometry analysis of FLG 29.1-sh7 and FLG 29.1-plKo cells. A representative panel is shown. MFI values from two independent experiments are reported in the text. (j) Western blots of pERK1/2 (top) and pAkt (bottom) of FLG 29.1-sh7, FLG 29.1-plKo and FLG 29.1 cells treated or not with 22 μm E4031 for 30 min. Membrane reprobing was performed as in b. Densitometric analysis is reported in Supplementary Figure S2. (k) Western blot of caspase 3 of FLG 29.1-sh7, FLG 29.1-plKo and FLG 29.1 cells treated or not with 22 μm E4031 for 30 min. Membrane reprobing was performed as in b. Densitometric analysis is reported in Supplementary Figure S2. (l) Percentage of Annexin V+/PI− cells in FLG 29.1-plkO and FLG 29.1-sh7 cells treated with 52 μm Cla for 48 h (P<0.01, Student’s t-test). (m) Percentage of Annexin V+/PI− cells in FLG 29.1-plkO cells treated with 52 μm Cla, or 78 μm Er, alone or in combination with 22 μM E4031. Values are mean±s.e.m. of two indipendent experiments each performed in triplicate.

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