Fig 2: CSA favours the formation of the FECH-CSA-CSB-RNAP1-RPS protein complex on native chromatin and its dissociation upon UV irradiation. (A) Immunoblot analysis with antibodies specific for FECH, HA, the catalytic subunit of RNAP1 (RNAP1_194), RPS10 and RPS15 in immunoprecipitations (IP) performed with anti-FECH antibodies or control IgG in the chromatin-enriched or TritonX100-soluble fractions (input) of CS3BE-wtCSAFlag-HA (+) or CS3BE-cassette1 (-) cells, before (-) and after 10 J/m2 UV irradiation (+). The amount of co-immunoprecipitated proteins was quantified and normalized to the amount of immunoprecipitated FECH and expressed in the diagram on the right as fold increase of the corresponding co-immunoprecipitated protein in CS3BE-wtCSAFlag-HA untreated cells. Reported values represent the mean ± SEM (n = 4), *P < 0.05, **P < 0.01 (Student's t-test). (B) Immunoblot analysis with antibodies specific for the catalytic and the RPA135 subunits of RNAP1 (RNAP1_194 and RNAP1_135, respectively), HA, FECH, RPS10, RPS15 and CSB in immunoprecipitations (IP) performed with anti-RNAP1_194 antibodies or control IgG in the chromatin-enriched fractions (input) of CS3BE-wtCSAFlag-HA (+) or CS3BE-cassette1 (-) cells, before (-) and after 10 J/m2 UV irradiation (+). The amount of co-immunoprecipitated proteins was quantified and normalized to the amount of immunoprecipitated RNAP1 and expressed in the diagram on the right as fold increase of the corresponding co-immunoprecipitated protein in CS3BE-wtCSAFlag-HA untreated cells. Reported values represent the mean ± SEM (n = 4), *P < 0.05, **P < 0.01 (Student's t-test). (C) Immunoblot analysis with antibodies specific for FECH, the catalytic subunit of RNAP1 (RNAP1_194), CSA, CSB, RPS15 and RPS10 in two-step co-immunoprecipitations (TIP) performed first by immunoprecipitating with anti-RNAP1_194 (2) antibodies and subsequently with anti-FECH (3) antibodies in the chromatin-enriched fractions (input) of primary dermal fibroblasts from an healthy donor. The immunoprecipitation with IgG (1) antibodies is a negative control. (D) Sub-cellular localization of FECH/RNAP1 interaction by proximity ligation assay (PLA) in CS3BE-wtCSAFlag-HA (+) and CS3BE-cassette1 (-) cells, before (-) and at 2 h after UV irradiation (10 J/m2). Red fluorescence signal visualizes the FECH/RNAP1 interaction. Nuclei were counterstained with DAPI. Scale bar: 10 µm.

Fig 3: Schematic depicting the contribution of RPS15 and IGF2BP1 to ESCC progression via the p38 MAPK pathway. a RPS15 overexpression promotes the proliferation and motility of ESCC cells. Mechanistic investigation revealed that RPS15 interacts with the KH domain of IGF2BP1, which directly binds and recognizes the MAPK14 and MKK6 mRNA 3′-UTR, and promotes translation of core p38 MAPK pathway proteins. b, RPS15 inhibition with folic acid treatment, IGF2BP1 ablation, or treatment with SB203580 suppresses ESCC proliferation and metastasis via the p38 MAPK signaling pathway

Fig 4: IGF2BP1 binds the 3′-UTR of MKK6 and MAPK14 in an m6A-dependent manner. a Distribution of m6A peaks of IGF2BP1 RIP-seq data across MAPK14 and MKK6 mRNA transcripts. b Enrichment of m6A modification in 3′-UTR region of MKK6 with Flag-tagged IGF2BP1 in KYSE30 cells (left); RIP-qPCR showing the binding of IGF2BP1 to the 3′-UTR region of MKK6 (right). c Enrichment of m6A modification in 3′-UTR region of MAPK14 with Flag-tagged IGF2BP1 in KYSE30 cells (left); RIP-qPCR showing the binding of IGF2BP1 to the 3′-UTR region of MAPK14 (right). d Agarose gel electrophoresis showing the binding of IGF2BP1 to the 3′-UTR region of MKK6 and MAPK14 in KYSE30 cells (left) and KYSE450 cells (right). e Western blot detected protein expression of the hallmark p38 MAPK-target gene set in RPS15-overexpressing KYSE30 cells (left) and KYSE450 cells (right) with or without IGF2BP1 knockdown. f Growth curves measured using Incucyte live-cell analyses of KYSE30 cells (upper) and KYSE450 cells (lower) stably transfected with control vector (blue) or RPS15-overexpressing vector (purple) and treated with IGF2BP1 knockdown or without IGF2BP1 knockdown for 72 h. Data were analyzed using unpaired t-tests. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 5: High RPS15 expression correlates with malignant phenotype and poor prognosis in ESCC. a Schematic created with BioRender.com demonstrating the screening strategy for pro-metastatic ribosome proteins (RPs). b, c qRT-PCR analysis of fold changes in gene expression of 89 RPs between metastatic and non-metastatic of KYSE30 cells (b) and KYSE450 cells (c). Data were presented as mean ± SD; n = 3. Two-tailed t-tests. d Venn diagram showing the RPs upregulated in metastasis of KYSE30 and KYSE450 cells. e qRT-PCR analysis of fold changes of four RPs expression in highly metastatic ESCC cell lines 30-D-4 and K450LM2. Data were presented as mean ± SD; n = 3. Two-tailed t-tests. f Representative images (left) and the statistical analysis (right) of TMA-based IHC showing higher RPS15 expression in tumor tissue (upper panel). The expression of RPS15 is correlated with lymph node metastasis (lower panel). Scale bars, 50 μm. The data were analyzed using paired two-tailed t-test and Fisher exact test and are presented as mean ± SD. *P < 0.05 and ***P < 0.001. g Kaplan–Meier survival curve showing that high expression of RPS15 correlated with lower disease-free survival in patients with ESCC (P < 0.05). h Multivariate Cox regression analysis of factors associated with survival of patients with ESCC