Fig. 4
KLF regulation of autophagy is conserved in mammalian cells. qPCR screens of HEK293 cells exposed to rapamycin treatment (a) and starvation (b). Cells were treated with respective regimen for 2 days prior to RNA isolation and qPCR analysis. Further details are given in Methods section. *P-value < 0.05 by Student’s T-test, N = 3 biological replicates. c Western blot analysis of LC3-I lipidation with and without KLF4 manipulation. Briefly, MEFs were treated with adenoviral Klf4 or siRNA targeting Klf4 and effects assessed by western blot 2 days afterwards. Data shown are representative of two independent experiments. Graphs represent quantitated LC3-II/LC3-I ratios. *P-value < 0.05 by Student’s T-test or after one-way analysis of variance followed by Tukey’s post hoc test. Further details in Methods section. d Autophagy pathway qPCR array analysis in MEFs with Ad-KLF4 or Si-KLF4 normalized to appropriate viral or siRNA control. MEFs were treated for 72 h prior to RNA isolation and qPCR analysis. Green oval represents significantly induced genes by Ad-KLF4 and blue oval represents genes reduced by Si-KLF4. P-value < 0.05 by Student’s T-test. N = 3 biological replicates. See also Supplementary Table 7 for full gene list of fold changes and P-values. e ChIP-qPCR in MEFs treated with control or adenoviral KLF4 of several target genes normalized to input DNA, then to nontarget control confirms KLF4 recruitment to CA/GCCC elements in regions upstream of autophagy genes. A locus upstream of GATA6 was used as a nontarget control. *P-value < 0.05 after one-way analysis of variance followed by the Dunnett’s post hoc test. N = 3 biological replicates. For list of ChIP-qPCR primers see Supplementary Table 9. All error bars represent standard error of the mean (SEM)
