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. 2025 Aug 29;11(35):eadv9401. doi: 10.1126/sciadv.adv9401

Fig. 5. hnRNPA 1 sustains ATF4 expression and stress responses.

Fig. 5.

(A) hnRNP A1 overexpression in 293FT cells rescues ATF4 protein expression suppressed by DKC1 knockdown. (B) hnRNP A1 overexpression restores cell proliferation suppressed by DKC1 knockdown. Cell growth data are presented as the means ± SEM (n = 4) and analyzed using a two-way ANOVA. (C and D) qRT-PCR (C) and immunoblotting (D) showing decreased mRNA and protein levels of amino acid metabolism–related genes following hnRNP A1 knockdown in the indicated neuroblastoma cell lines. qRT-PCR data are presented as the means ± SEM (n = 4) and analyzed by a two-way ANOVA. (E) Immunoblotting showing that hnRNP A1 knockdown abrogates stress-induced up-regulation of ATF4 and its downstream amino acid metabolism targets in BE(2)-C and SHEP1 cells. HisOH, AAR inducer; Tm, ER stress inducer. (F and G) hnRNP A1 binds ATF4 mRNA: (F) immunoblotting of hnRNP A1 in control (IgG) and anti–hnRNP A1 immunoprecipitates; (G) qRT-PCR analysis of ATF4 and ACTIN mRNAs in the corresponding immunoprecipitates. Data are shown as the means ± SEM (n = 2) and analyzed by a two-way ANOVA. (H) hnRNP A1 knockdown reduces ATF4 mRNA stability in BE(2)-C cells. RNA was collected at the indicated time points following treatment with actinomycin D (5 μg/ml) and analyzed by qRT-PCR. ATF4 mRNA levels were normalized to B2M and presented as a fraction of the initial value at time zero. h, hours. Data are shown as the means ± SEM (n = 3). ***P < 0.001; ns, not significant.