Fig 2: Silencing of B5 prevents HSV ICP4 transcripts from associating with ribosomes.(A). Cytoplasmic extracts of CaSki cells transfected 48 h earlier with siHVEM or siB5 were fractionated on 10% to 50% sucrose gradients and the UV absorbance at 254 nm of each fraction (bottom to top of the sucrose gradient) measured; the relative positions of 40S and 60S ribosomal subunits, 80S monosomes and polysomes are indicated (left). The RNA from each fraction was analyzed for GAPDH expression by reverse transcription and PCR. Alternatively, fractions were analyzed for expression of PABP and B5 by Western blot (right). (B). Cytoplasmic extracts from HSV-2 infected cells that had been transfected 48 h earlier with siHVEM or siB5 were fractionated on 10% to 50% sucrose gradients and the UV absorbance at 254 nm of each fraction measured (left). The RNA from each fraction was analyzed for GAPDH or ICP4 expression by reverse transcription and PCR and the expression of ICP4 protein by Western blot (right).

Fig 3: Biomechanical imaging of HeLa cells. The enhanced contrast enabled the acquisition of Brillouin images of single cells at different depths (?z = 1 µm) in the case of uninduced (a) and doxycycline-induced (b) cells. Fluorescent images (c–d) showing the PABP (green) and DNA (blue; labeled by DAPI) merged staining. e–f Associated DIC images. Scale bar, 10 µm. g Box-and-whisker plot of the Brillouin frequency shift for different cellular compartments with (+) and without (-) FUSP525L expression. In both cases the expression of mutant FUS did not significantly altered the cytoplasm (Student’s t-test p = 0.22, N = 25 cells) and the nucleus (p = 0.37) properties (h) Box-and-whisker plot of the Brillouin frequency shift of living (-) and fixed (+) HeLa cells (N = 29, 3 experiments). Despite an overall increase in Brillouin frequency, different subcellular compartments are found to be similarly altered in living and fixed cells as indicated by the frequency ratio q

Fig 4: Ectopic expression of influenza A virus PA and NP ORFs inhibits SG formation induced by arsenite and pateamine A.HeLa-TetOff cells were transfected with the expression vectors for the indicated c-terminally myc-tagged IAV ORFs or the EGFP or mRFP expression vectors and treated with sodium arsenite or PatA 24 hours post-transfection. 50 min post-treatment SG formation was analyzed by immunofluorescent staining for TIA-1 or TIAR and SG co-localization of viral ORF-encoded proteins by staining for myc epitope. (A). Arsenite-induced SG formation (dark grey bars) and SG recruitment (double-positive SGs, light grey bar overlays) was quantified from 3 independent experiments. (B). Representative immunofluorescence images demonstrating arsenite-induced SG inhibition by PA-myc and NP-myc (arrows), SG co-localization of M1-myc and NS1-myc proteins (circles), and nuclear PABP1 relocalization in PA-myc expressing cells. (C). PatA-induced SG formation (dark grey bars) and SG recruitment (double-positive SGs, light grey bar overlays) in cells expressing the indicated IAV ORFs and the control mRFP protein was quantified from 2 independent experiments. (D). Representative immunofluorescence images demonstrating SG inhibition by PA-myc and NP-myc (arrows) in response to PatA treatment, and SG co-localization of NS1-myc protein (circles).

Fig 5: Model of YB-1's involvement in tiRNA-mediated stress response pathway. (A) Actively translating mRNA is circularized through the interaction of eIF4F (orange), PABP (blue), translation termination factors eRF1/eRF3 (pink). (B) During stress, tRNAs are cleaved by ANG creating 5'-tiRNAs which displace eIF4F from m7GTP cap of mRNA (C), thereby inhibiting translation initiation. (D) YB-1 (green-orange) acts to package inactive mRNPs, inhibited through the action of 5'-tiRNAs, into SGs.