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. Author manuscript; available in PMC: 2013 Apr 17.
Published in final edited form as: Cancer Cell. 2012 Apr 17;21(4):532–546. doi: 10.1016/j.ccr.2012.02.019

Figure 6. MAP1LC3B is a direct target of miR-204.

Figure 6

(A) Sequence of the wild-type (WT) miR-204 site in the 3’UTR of LC3B, and mutation (mut) of the core binding site (a). (b) The 3’UTR of LC3B with the wild-type but not mutant miR-204 site confers regulation by miR-204 to the luciferase reporter construct.

(B) Effects of an anti–miR-204 lentiviral Zip or siRNAs on LC3B protein expression in 786-O VHL(+) RCC cells shown by western blot and quantification of 3 independent experiments.

(C) Regression analysis showing negative correlation between normalized miR-204 level and normalized LC3B protein level in human ccRCC tumors with the wild-type VHL, performed as described in Figure 2D.

(D) Representative western blot showing tumors with high and low levels of LC3B. Normalized values of miR-204 levels for each tumor are shown in a graph below the blot. Expression of LC3B in 786-O cells is shown as reference.

(E) Western blot showing accumulation of LC3B-II in 786-O VHL(+) cells treated with anti-miR-204 in the absence or presence of 100 μM chloroquine.

(F) 786-O VHL(−) cells transfected with RFP-LC3B or RFP were sorted, plated, and maintained for 48 hr in medium containing 0.1% serum. Cells were then treated with 25 nM short pre-miR-204 or scramble control construct and, 48 hr later, stained by DAPI. Fraction of live cells was determined among the stained and unstained cells in both conditions.

(G) Quantification of endogenous ATG16L puncta per cell in 786-O VHL(−) cells transfected with LC3B-RFP or RFP alone and treated with miR-204 as in (F). Red cells: (+) indicates cells expressing RFP fluorescence from either construct; (−) indicates cells that did not express RFP fluorescence. Numbers of cells (n) in which puncta were counted is shown below the graph.

(H) Effect of LC3B knockdown on cell death induced by miR-204. 786-O or A498 VHL(−) cells stably transfected with two different LC3B shRNAs or scramble construct were additionally transfected with miR-204 or scramble control. Two independent pools of 786-O cells expressing LC3B sh I RNA were used (Pool 1 and Pool 2). N, non-treated cells; L, cells transfected with Lipofectamine only; Scr, cells transfected with scramble pre-miR-204 control; 204, cells transfected with 50 nM of pre-miR-204. See also Figure S6.