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PNAS 108 (43): 17749-17754

Copyright © 2011 by the National Academy of Sciences.

A role for c-Myc in regulating anti-mycobacterial responses

Howard C. H. Yim, James C. B. Li, John C. H. Pong, and Allan S. Y. Lau1

Cytokine Biology Group, Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China


Figure 01
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Fig. 1.. Mycobacteria induce Myc expression. PBMac were treated with (A) mock or BCG [multiplicity of infection (M.O.I.) = 1], (B) mock or M. avium (M.O.I. = 20), and (C) mock or M. chelonae (M.O.I. = 10) or M. kansasii (M.O.I. = 1) for the indicated time. (D) PBMac were treated with mock or BCG with the indicated M.O.I. for 24 h. (AD) Cellular proteins were extracted for Western blots to analyze the levels of Myc and Actin. The data are representative results from cells isolated from three independent blood donors. The values under each lane represent the fold induction of Myc versus 0 h or the unteated cells after normalization to the level of Actin. (E) PBMac were treated with mock or BCG (M.O.I. = 1) for the indicated time. (F) PBMac were pretreated with DMSO, 10μM inhibitors antagonizing ERK1/2 (PD), JNK (SP) or p38 MAPK (SB), or 15μg/mL inhibitor against NF{kappa}B (CAPE) for 1 h, and followed by treatment with mock or BCG (M.O.I. = 1) for 3 h. (E and F) Total RNA was extracted for analyzing the mRNA expression levels by quantitative RT-PCR. (G) PBMac were treated with mock or BCG (M.O.I = 1) for 24 h. PBMac and THP-1 cells were fixed and stained with propidium iodide. The DNA contents of the cells were analyzed by flow cytometry and ModFit 3.2 software. (EG) The data are presented as mean ± SEM from three independent blood donors. * denotes P < 0.05 as determined by Student's t test.

 

Figure 02
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Fig. 2.. Myc localizes in the cytoplasm. PBMac were treated with mock or BCG (M.O.I. = 1) for 5 and 24 hours. Cells were fixed and labeled with monoclonal primary antibody against Myc (4A6, Millipore; A) or polyclonal primary antibodies against Myc (N262, Santa Cruz Biotechnology; B), and followed by the corresponding secondary antibodies. Subsequently, cell nuclei were labeled with DAPI. Images of cells were analyzed by Cellomics ArrayScan HCS VTI System. The cytoplasmic localization of Myc was quantified and expressed as nuclear/cytoplasmic intensity ratio (ratio > 1 implies in nucleus, ratio < 1 implies in cytoplasm). The data are presented as mean ± SEM from three independent blood donors.

 

Figure 03
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Fig. 3.. Myc specifically mediates mycobacteria-induction of cytokines and suppresses the intracellular growth of mycobacteria. (AE) PBMac were transfected with control or Myc siRNA-a (AC) or control or Myc siRNA-b (D and E) for 48 h, and followed by treatment with mock or BCG (M.O.I. = 1) for 24 h. Supernatants were harvested for determining the levels of the indicated cytokines by ELISA. (F and G) PBMac were treated with control or Myc siRNA-a (F) or control or Myc siRNA-b (G) for 48 h and followed by treatment with M. avium (M.O.I. = 20) for 8 h. Cells were either lysed for determining the number of intracellular bacteria or were washed with PBS. After washings and treatment with trypsin/EDTA, the cells were incubated with fresh culture medium for another 40 or 88 h. At the indicated time, cells were then lysed for determining the number of intracellular bacteria. The numbers of intracellular M. avium bacteria in cells transfected with Myc siRNA-a or Myc siRNA-b cells were expressed as fold induction over those transfected with control siRNA-a or control siRNA-b, respectively. The data are expressed as mean ± SEM from 8 (A), 7 (B), 3 (C), 4 (D), 4 (E), 3 (F), and 10 (G) independent blood donors. For AE, * and n.s. denote P < 0.05 and P > 0.05, respectively, as determined by Student's t test. For F and G, * denotes P < 0.05 as determined by two-way ANOVA.

 

Figure 04
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Fig. 4.. Myc augments mycobacteria-activation of IRAK1, MAPK and NF{kappa}B. PBMac were transfected with control or Myc siRNA-a as in Fig. 3 AC for AE, and with control or Myc siRNA-b as in Fig. 3 D and E for FJ before BCG (M.O.I. = 1) treatment for the indicated time. Cytoplasmic proteins were analyzed by Western blot. (A and F) The protein level of IRAK1 was quantified, normalized with that of Actin, and expressed as fold change over the treatment with control siRNA-a or siRNA-b and mock. (BD and GI) The levels of phosphorylated proteins of EKR1/2, p38 kinase and Akt at each time point were normalized with the level of their respective total proteins and expressed as fold induction over their basal levels at 0 h. (E and J) The protein level of I{kappa}Bα was normalized with that of Actin at each time point and expressed as fold change over its basal level at 0 h. The data are expressed as mean ± SEM from three (A), five (B), four (C), three (D), four (E), four (F), four (G), three (H), three (I), and three (J) independent blood donors. M denotes mock. * denotes P < 0.05 as determined by Student's t test.

 

Figure 05
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Fig. 5.. Overexpression of Myc is sufficient for TNF-α induction and IRAK1 activation. PBMac were electroporated with the indicated expression plasmid and incubated with culture medium for 48 h. Supernatants were discarded and the cells were incubated with fresh culture medium for 24 h. (A and B) Supernatants were harvested for determining the levels of the indicated cytokines by ELISA. The data are expressed as mean ± SEM from four (A) and three (B) independent blood donors. (C and E) Total proteins were harvested for analysis by Western blot. The data are representative results from cells isolated from three independent blood donors. (D and F) Protein levels of Myc (D) and IRAK1 (F) were normalized with that of Actin and expressed as fold induction relative to the cells electroporated with pcDNA3.0 plasmid. The data are expressed as mean ± SEM from three independent blood donors. The * and n.s. denote P < 0.05 and P > 0.05, respectively, as determined by Student's t test. The # denotes nonspecific bands.

 


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