JCS038208 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Staufen knockdown facilitates stress granule formation. U2OS cells were treated with the indicated siRNAs, exposed to arsenite and stress-granule-positive cells were identified by TIA-1 staining. (A) At least 300 cells were counted from duplicate coverslips for each point, ***P<0.0001 for 60, 90, 120 and 180 minutes. (B) Immunostaining for the stress granule and processing body markers TIA-1 and Dcp1a, at the 90 minute time point as shown in A. Processing bodies are reduced in size and number in both control and stress conditions in siHedls- or siRck-treated cells. Stress granule formation is moderately impaired upon knockdown of these processing body components. Scale bar: 1 µm. (C) Representative immunofluorescence of the 2 hour time-point shown in figure 3G. Percentage of stress-granule-positive cells is indicated.

• Supplemental Figure S2 -

  Fig. S2. (A) NIH 3T3 or BHK cells were transfected with Stau1-V5 and Stau1-ECFP, respectively. stress granules were induced with arsenite 8 hours after transfection and immunostaining for the indicated molecules was performed. Scale bar: 10 µm. (B) NIH 3T3 cells were transfected with ECFP or Stau1-ECFP, exposed to oxidative stress and stained for Stau1 and TIAR. Overexpresion levels were determined in single cells in the range where the Stau1 immunofluorescence intensity was lineal with the fluorescence intensity of the ECFP moiety, which is directly proportional to the expression level of the construct. Stress granule formation index and overexpression levels are indicated. (C) Cells were transfected with the indicated constructs and stress granule formation upon oxidative stress induction was evaluated 48 hours after transfection. Stau1-ECFP specifically impairs stress granule formation.

• Supplemental Figure S3 -

  Fig. S3. Cells were transfected with Stau1-ECFP, exposed to arsenite and immunostained for phosphorylated eIF2α and P0 as a control staining. As expected, immunofluorescence signal for phosphorylated eIF2α was negligible in resting conditions and detected both dispersed and in association with stress granules (arrowheads) upon oxidative stress. Induction of phosphorylated eIF2α was similar in Staufen 1 transfected cells and non-transfected cells, with or without stress granules. Bottom, treatment with cicloheximide simultaneous to the stress stimuli abrogates stress granule formation thus allowing comparison of non-punctate P-eIF2α signal in transfected and non-transfected cells. Induction of eIF2α phosphorylation upon stress was similar in all cases.

• Supplemental Figure S4 -

  Fig. S4. NIH 3T3 cells were treated with the indicated siRNAs and pulsed with 250 nM TG or 0.25 mM arsenite for 1 hour. Protein synthesis was evaluated by incorporation of radiolabeled aminoacids after a 15 min pulse performed at the indicated time points after stimulation. Percentage of stress granule-positive cells and the stimulation of stress granule formation upon Stau1 knockdown (siStau1/siNR) is indicated. No significant differences in the inhibition of protein synthesis that temporally correlates with stress granule formation were observed, whereas stress granule formation was facilitated by Stau1 knockdown. Similarly, the partial recovery of protein synthesis that correlates with stress granule dissolution and translation of HSP70 (data not shown) was not affected by Stau1 depletion.