Fig 2: SRSF3 interacts with eIF4A1 in the cytoplasm.A The siControl or siSRSF3 treated SW480 cells were detected SRSF3, p21cip1/waf1 and translation initiation factor proteins. The protein, β-actin was used as a loading control. B HEK-293T cells transfected with Flag-eIF2α, Flag-eIF5A, Flag-eIF3F, and Flag-eIF4G1. Co-immunoprecipitation was carried out using the anti-Flag antibody or eIF4A1 antibody. Levels of indicated proteins in cytoplasmic extract and Co-IP products were analyzed by western blot analysis. C Cytoplasmic extract of SW480 cells were immunoprecipitation with an anti-SRSF3 or anti-eIF4A1 antibody. The immunocomplexes and input were analyzed using western blot with anti-Flag, anti-SRSF3, anti-rpS3, and anti-rpL13a antibodies. D U2OS cells were treatment with or without sodium arsenite (0.5 mM, 1 h) for the mentioned time period before for immunofluorescence microscopy. Cells were stained with eIF4A1 (Green), SRSF3 (Red). Scale bar, 10 μm. E Schematic representation of full length and a series of deletion mutants of SRSF3. F Lysates from HEK293T cells transfected with Flag-tagged full length (FL) or deletion mutant SRSF3 were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis with anti-Flag, anti-eIF4A, and anti-β-actin antibodies. G In vitro GST-pulldown assay of the binding of recombinant Hisx6-tagged eIF4A1 with GST or the GST-SRSF3 (FL, RRM, and RS domain).

Fig 3: Depletion of eIF4E phosphorylation engenders inflammatory and depression-like phenotypes via selective translational control of a subset of mRNAs. A, Ablation of the single phosphorylation site on eIF4E (Ser209→Ala) does not affect global protein synthesis, but rather the translation of a subset of mRNAs harboring GAIT elements, which engenders a depression-like phenotype in 4Eki mice. 4Eki mice also display increased expression of inflammatory cytokines, which could be linked to disinhibition of GAIT translational repression and possibly to depression-like phenotypes. Altered cap binding of the helicase eIF4A1 and/or of the GAIT complex protein rpL13a could be the mechanism underlying altered translation initiation following depletion of Ser209 eIF4E phosphorylation. B, The SSRI fluoxetine requires eIF4E phosphorylation to exert its antidepressant action. C, Phosphorylation of eIF4E promotes anti-inflammatory and antidepressant pathways.

Fig 4: PAX2 induces apoptosis via IRF1.a Western blotting showing induction of caspase 7 and 8 level by PAX2. MCF-7-PAX2 cells were treated with vehicle (Veh), 50 ng/ml doxycycline (Dox), 1 μM tamoxifen (Tam) or the combination of both for 48 h, then whole cell lysate was prepared and analyzed by using western blotting with caspase 7, caspase 8, and RPL13a antibodies, RPL13a is used as loading control. Cleaved caspase 7 was quantified normalization against RPL13a. b MCF-7-PAX2 cells were transfected with siNT or siIRF1 for 24 h, then treated with or without 50 ng/ml doxycycline (Dox) for 48 h. Whole cell lysate was prepared and analyzed with western blotting with caspase 7, caspase 8, and RPL13a antibodies. c, d Real-time Annexin V assay showing PS externalization. MCF-7-PAX2 cells were transfected with siNT, or siIRF1 followed by treatments with vehicle (Veh), 50 ng/ml doxycycline (Dox), 1 μM tamoxifen (Tam), or the combination of both. PS externalization was measured according to the methods session. The data are represented as the mean of independent replicates ± s.d. (n = 5). e The model of actions of PAX2 in ER-positive breast cancer cells.

Fig 5: The gene sets of lactate responsive-, neuronal differentiation-related genes in undifferentiated and differentiated SH-SY5Y cells.A, Venn diagram of upregulated genes upregulated by lactate treatment shared with SH-SY5Y and Neuro2A. B, the effects of lactate treatment on mRNA expression of neuronal differentiation-related genes in SH-SY5Y (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, versus control. Statistical significance was determined by one-way ANOVA followed by Dunnett tests. C, the effects of 30 mM lactate on mRNA expression of neuronal differentiation-related genes during differentiation of SH-SY5Y (n = 3). ∗p < 0.05 versus control at the same time point. Statistical significance was determined by one-way ANOVA followed by Dunnett tests. p-value versus control during differentiation was determined by two-way ANOVA followed by Sidak's multiple comparison tests. D, the effects of 30 mM lactate on mRNA expression of neuronal differentiation-related genes in primary embryonic mouse neuronal cells (n = 4). ∗p < 0.05, versus control. Statistical significance was determined by an unpaired two-tailed t test. Following densitometric quantification, each gene expression values were normalized to corresponding values of RPL13A for human or Rpl13a for mouse and presented as fold change to control. All data are reported as means ± SEM.