Journal: Nucleic Acids Research
Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay
doi: 10.1093/nar/gkag075
Figure Lengend Snippet: Poly(A)-binding proteins PABPC1 and PABPC4 are necessary for PUM-mediated repression. ( A ) Western blot analysis confirms auxin induced depletion of degron-tagged PABPC1-AID and RNAi depletion of PABPC4 either individually or in combination in HCT116 cells. RNAi was performed by transfecting cells with either PABPC4 or NTC siRNAs. PABPC1-AID protein was depleted upon treatment with indole-3-acetic acid (+IAA), in comparison to vehicle only control (−IAA). GAPDH served as a loading control. ( B )PUM-mediated repression of Nluc 3×PRE reporter expression was measured in HCT116 cells, relative to the mutant PRE version. Individual and combined effects of PABPC4 RNAi and IAA-induced depletion of PABPC1-AID on PUM repression were tested. n = 9; three experiments, each with three biological replicates; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, **** P < .001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( C ) Western blot analysis of samples from a representative experimental replicate for experiments shown in panels ( D ) and ( E ). GAPDH served as a loading control and PUM1 and PUM2 protein levels were detected as controls. The effect of combined PABPC4 RNAi and PABPC1-AID depletion on PUM repression of Nluc reporters containing the 3′ UTRs of the natural, PRE-containing, PUM target mRNAs FZD8 in panel (D) and CDKN1B in panel (E) were measured, relative to matched PRE mutant versions. n = 9; three experiments, each with three biological replicates; ± SD. ( F ) Live cell numbers were measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD. ( G ) Cell viability was measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD.
Article Snippet: After 6 h, fresh media containing 1 μg/ml of doxycycline and 0.5 mM of indole-3-acetic acid (IAA, Goldbio, I-110–25) were added to the test samples.
Techniques: Binding Assay, Western Blot, Comparison, Control, Expressing, Mutagenesis
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