Fig 2: Involvement of RACK1 in NHE6 relocalization to the plasma membrane.(a) Co-immunoprecipitation of endogenous RACK1 with HA-tagged NHE6 from transiently transfected HT-1080 cells incubated under normoxia or hypoxia for the indicated times. Data represent 5% of the total cell extract used for each immunoprecipitation. (b) Immunoblot analysis of HT-1080 cell lysates 48 h after transfection with non-targeting siRNA or RACK1-specific siRNA. α-tubulin was used as a loading control. The blots shown in (a) and (b) are representative of four and three independent experiments, respectively. (c and d) Quantification of NHE6-GFP at the (c) plasma membrane and at (d) EEA1-positive endosomes in HT-1080 cells transfected with scrambled siRNA or RACK1-specific siRNA (n=3–4 independent experiments and >75 cells per experimental condition). (e) Endosomal–lysosomal pH measured in HT-1080 cells in the presence of non-targeting siRNA or RACK1-specific siRNA (n=3 independent experiments with >75 cells per experimental condition). (f and g) Percentage of Dox fluorescence within (f) DAPI-stained nucleus and (g) Tfn-labelled endosomes of HT-1080 cells (n=3–4 independent experiments and >75 cells per experimental condition). Bars represent the mean±s.e.m. (**P<0.01, unpaired Student’s t-test).

Fig 3: Regulation of PDE4D activity at IFNAR2 by TPL2. (a) T24 cells were treated with IFN-α (1 × 104 U/mL) for specific time points. The levels of phosphorylated and total TPL2 bound to IFNAR2 or IFNAR1 were detected by performing western blotting after co-immunoprecipitation using IFNAR2 or IFNAR1 antibodies. (b) T24 cells were treated with IFN-α (1 × 104 U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. The levels of phosphorylated and total TPL2 bound to IFNAR2 or IFNAR1 were detected by performing western blotting after co-immunoprecipitation using IFNAR2 or IFNAR1 antibodies. (c) T24 cells were treated with IFN-α (1 × 104 U/mL) for specific time points. The levels of RACK1 and PDE4D that interacted with IFNAR2 or IFNAR1 were detected after co-immunoprecipitation using IFNAR2 or IFNAR1 antibodies. (d) T24 cells were treated with IFN-α (1 × 104 U/mL) for specific time points. The intracellular cAMP level, activity of total PDE4D, and activity of PDE4D that interacted with IFNAR2 were detected after co-immunoprecipitation using IFNAR2 or PDE4D antibodies. (e) T24 cells were treated with IFN-α (1 × 104 U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. The activity of total PDE4D was detected after immunoprecipitation using PDE4D antibody. (f) T24 cells were treated with IFN-α (1 × 104 U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. The PDE4D that interacted with IFNAR2 and their activity were detected after co-immunoprecipitation using IFNAR2 antibody. Data represent the results of five independent experiments. Error bars indicate mean ± SD. *, P < 0.05; **, P < 0.01 (t-test)

Fig 4: Knockdown of RACK1 inhibited an ongoing PRRSV infection. Marc-145 cells were infected with PRRSV for 12 hours to allow the establishment of the viral infection, followed by siRNA treatments. The mRNA and protein expression level of cellular RACK1 and viral ORF7 were analyzed at different time points after transfection (48, 72 and 84 hours post transfection). (A) Cellular RACK1 mRNA expression level. (B) Absolute copy number of viral ORF7 mRNA. (C) Western blot of cellular RACK1 and viral N protein level. GAPDH was used as internal control in both RT-qPCR and western blot analysis. These data is representative for three independent experiments.

Fig 5: RACK1 suppresses CCNO expression through enhancement of miR-302b-3p, miR-302c -3p or miR-302d-3p expression. a The expression of RACK1, miR-302b/c/d-3p, and CCNO in cells detected by RT-qPCR, *p < 0.05 vs. cells treated with oe-NC; b Protein levels of RACK1 and CCNO examined by Western blot analysis, with protein bands assessed, *p < 0.05 vs. cells treated with oe-NC; c The correlation between RACK1 and miR-302b/c/d-3p in CasKi cells analyzed by Pearson’s correlation coefficient; d Positive expression of RACK1 protein in CSCC (N = 46) and normal cervical tissues (N = 30) detected by immunohistochemistry (×400). *p < 0.05 vs. cells treated with oe-NC. The values in the figure were measurement data, and expressed as mean ± standard deviation. Independent sample t test was used for data comparison between two groups. The experiment was repeated 3 times independently