TAK1 Regulates Autophagic Cell Death by Suppressing the Phosphorylation of P70 S6 Kinase

TAK1 regulates autophagic cell death by suppressing the phosphorylation of p70 S6 kinase1

Ju Hyun Shin1,3, Sang-Hyun Min1,5, Seong-Jin Kim3, Young-Il Kim1, Junsoo Park4 , Heung Kyu Lee*,2, and Ook Joon Yoo*, 2

Supplementaryinformation

Supplementary materials and methods

Flow cytometric analysis

HEK 293T cells were transfected and incubated as indicated in the figure legends. Cells were harvested and resuspended in PBS. Samples were transferred into FACS tubes and centrifuged. Harvested cell pellets were resuspended in 0.5 ml of cold PBS1. Analysis of GFP fluorescence intensity was performed with a BD FACSCalibur Flow Cytometer (BD, San Jose, CA, USA).

Generation of S6K1 mutant construct

cDNA for human S6K1 delta CT (corresponding to S6K1 amino acids 1-398) was prepared by PCR mutagenesis and subcloned into pCMV-Myc vector (Clontech, Mountain View, CA, USA).

Supplementary figures and figure legends

Supplementary Figure 1. Autophagy-related proteins are involved in TAK1-induced cell death.

HEK 293T cells were transfected with 100 nM of the indicated siRNAs for 16 h and transfected with TAK1 for another 24 h. Cell viability was analysed by trypan blue exclusion assay.The values represent the means ± s.d. of three independent experiments. * P < 0.05.

Supplementary Figure 2.TAK1-induced autophagy is verified by FACS analyses.

(A) and (B) HEK 293T cells were co-transfected with GFP-LC3 and indicated plasmids for 48h. As a positive control, cells were treated with rapamycin (10μM) for 4 h. Cells were harvested and analysed by FACS.

Supplementary Figure 3. TAK1 induces autophagy in various conditions.

(A) GFP-LC3 was co-transfected with designated plasmids. After transfection, cells were incubated for 48 h in normal culture media and harvested for immunoblotting.

(B) 48 h after transfection, cells were treated with rapamycin (10μM) for 4 h or

Supplementary Figure 4.TAK1 influences phosphorylation of S6K1.

(A) HEK 293T cells were transfected with the indicated plasmids or 100 nM of TAK1 siRNA. The expression levels of TAK1, endogenous LC3-I, LC3-II, p-p70S6K1and β-Actin were analysed by immunoblotting. (B)48 h after transfection, cells were treated with IL1-(20 ng/μl) for 30 min.(C) p-p70S6K1 level was quantified using the Adobe Photoshop tool.

Supplementary Figure 5.S6K1 C-terminal deletion mutant (Myc-S6K1 delta CT) shows reduced TAK1-binding intensity.

HEK293T cells were tranfected with the indicated genes, and 48h after transfection, cell lysates were immunoprecipitated with an anti-Myc antibody. The blots in the lower three panels were obtained from the same cell lysates using the indicated antibodies.

Supplementary Figure 6.TAK1 kinase activity affects TAK1-S6K1 binding.

Myc-S6K1 was co-transfected with Flag-tagged TAK1-WT or TAK1-KWand incubated for 48 h. Cells were immunoprecipitated with an anti-Myc antibody and then immunoblotted with the indicated antibodies.

Supplementary Figure 7.Kinase inactive form of TAK1 hardly has an influence on the raptor-S6K1 binding.

Cells were transfected with the indicated constructs and incubated for 48 h. Cell lysates were immunoprecipitated with an anti-HA antibody and collected for immunoblotting with the indicated antibodies.

Supplementary Figure 8.TAK1 induces cytotoxic autophagy in HEK 293T cells.

HEK 293T cells were transfected with indicated constructs for 24 h and were analysed for cytotoxicity with a LDH release assay. The error bars represent mean ± s.d. for three independent experiments. * P < 0.05 and ** P < 0.01

Supplemental Reference

1.Shvets, E. Elazar, Z. Flow cytometric analysis of autophagy in living mammalian cells. Methods Enzymol452, 131 (2009).