Fig. 2: HuB promotes the expression of proinflammatory genes.

A HuB enhances HuR binding with inflammatory genes’ mRNAs. HEK293 cells were transfered with siRNA targeting HuB or with control siRNA, and then mock-treated or exposed to TNFα (10 ng/mL) for 1 h. The cytosolic lysates were collected and RNA-IP was conducted using HuR antibody. Half of the bead-antibody-protein/mRNA complexes were utilized for western blotting to assess equal loading/input of HuR, and the remaining half was subjected to real-time PCR to detect the levels of HuR antibody-precipitated TNFα, CXCL1, and CXCL2 mRNAs via normalization to the mRNA levels detected from the whole-cell lysates as the input. The levels of mRNAs precipitated from the group transfected with control siRNA and absent of TNFα treatment were set as 1, to normalize the mRNA levels of other samples. B Biotin-labeled tandem ARE repeat-containing RNA oligonucleotides. C Both HuB and HuR bind with ARE-containing RNA. GST, GST-HuB, and GST-HuR were incubated with biotin-labeled tandem ARE repeat-containing RNA oligos as indicated. The gel retardation assay was performed to detect the binding of the recombinant proteins to the probes. D Biotin-labeled TNFα mRNA’s 3’UTR ARE repeat-containing RNA oligonucleotides. E Both HuB and HuR bind with TNFα mRNA’s ARE. GST, GST-HuB and GST-HuR were purified and eluted, and then incubated with TNFα ARE. The gel retardation assay was performed to detect the binding of the recombinant proteins to probes. F HuR and HuB bind to the same RNA sequence as a single complex. GST-HuB and GST-HuR were purified and eluted, and then incubated with TNFα ARE. The gel retardation assay was performed to detect the binding of the recombinant proteins to probes. G, H HuB knockdown mitigates the expression of inflammatory genes. HEK293 cells were subjected to siRNAs targeting HuB and then mock treated or exposed to TNFα (10 ng/mL) for 1 h; Western blotting shows the efficacy of HuB knockdown; Real-time PCR was performed to detect the mRNA expression of TNFα, CXCL1 and CXCL2 (G). The level of TNFα protein was detected by western blotting. Quantitative assessment of the relative levels of TNFα and HuB was achieved via western blotting and normalized to that of β-actin (H). I HuB knockdown decreases proinflammatory mediators’ mRNA half-lives in TNFα exposed cells. HEK293 cells were subjected to siRNA targeting HuB followed by being mock-treated or exposed to TNFα (10 ng/mL) for 1 h to boost the transcription of proinflammatory mediators, and then subjected to transcriptional inhibition with or without TNFα maintenance for various times as indicated; Western blotting shows the efficacy of HuB knockdown. Quantitative assessment of the relative levels of HuB was achieved via normalization to that of β-actin. Real-time PCR was performed to assess the remaining mRNA levels of TNFα. Data were normalized to β-actin mRNA, and the relative amount of mRNA without Act D treatment was taken as 100%. Quantification shows mean ± SD based on three independent experiments, with significance of the difference determined by one-way (A, G, and H) or two way (I) ANOVA test. n = 3 independent experiments (A–I), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are available in supplementary data 1 for this figure.