Fig 2: Tumor suppressive functions of RINES in carcinoma cells. (A) Schematic representation of full‐length RINES (isoform 1), RINES‐isoform 2, and the RING domain‐deleted mutant (RINES‐ΔRING). CCD, coiled‐coil domain; TM, transmembrane domain. RINES protein expression is shown in a normal tissue panel and in paired adjacent normal and tumor tissues. (B) Subcellular localization assay demonstrated co‐localization of RINES with the ER. HONE1 cells were co‐transfected with RINES, isoform 2, or RING‐mutant plasmids along with an ER‐specific marker (pDsRed‐ER). Nuclei were counterstained with DAPI (blue). Original magnification, 400×. Scale bar 100 µm. (C) Representative colony formation assay performed in monolayer cultures of tumor cells expressing RINES or isoform 2. Quantitative analysis of the colony numbers is shown, with data presented as mean ± SD from three independent experiments using one‐way ANOVA with Tukey's post hoc test. Colonies with more than 50 cells in empty vector‐transfected cells were designated as 100 for comparison. ****, p < 0.0001. (D) Cell viability of tumor cells expressing RINES or isoform 2 was assessed using the CCK‐8 assay. Data are presented as mean ± SD from three independent experiments using one‐way ANOVA with Tukey's post hoc test. ***, p < 0.001. (E) RINES suppresses tumor formation in the nude mice model of ESCC. KYSE150 cells infected with a CMV‐Firefly luciferase lentivirus vector encoding RINES or an empty vector were subcutaneously injected into BALB/c nude mice. Tumor progression was monitored by measuring body weight, tumor weight, and bioluminescence intensity. Data are presented as mean ± SD with representative images shown. Statistical significance was determined using an unpaired Student's t‐test. *, p < 0.05. (F) TUNEL assay results in KYSE510 and HCT116 cells. Red fluorescence signals indicate TUNEL‐positive (apoptotic) cells. Data are presented as mean ± SD of three independent experiments using one‐way ANOVA with Tukey's post hoc test and representative data are shown. Scale bar, 50 µm. (G, H) Matrigel chamber assays evaluating migration (G) and invasion (H) of tumor cells expressing RINES or isoform 2. Data are represented as mean ± SD of three independent experiments by one‐way ANOVA with Tukey's post hoc test, and representative data are shown. Scale bar, 50 µm. (I) RINES, but not isoform 2, inhibits lamellipodia formation in MB231 cells. Scale bar 100 µm. (J) Western blot detection of markers for proliferation (Aurora A, PCNA), apoptosis (cleaved caspase‐3, cleaved PARP), and EMT (E‐cadherin, vimentin) in carcinoma cell lines.

Fig 3: Increased STAT3 and MYC expression rescues RINES‐induced stemness suppression. (A, B) Tumor spheroid formation was assessed in RINES‐expressing KYSE510 (A) and NPC43 (B) cells transfected with GFP‐STAT3 or GFP‐MYC. Representative images of tumor spheres were captured after 7 days of culture. Graphs show the quantitative analysis of sphere‐forming efficiency and size distribution. Data are presented as mean ± SD of three independent experiments. Statistical significance was determined by one‐way ANOVA with Tukey's post hoc test. ****, p < 0.0001. Scale bar, 50 µm. (C, D) Western blot analysis of stemness regulators and stem cell markers in RINES‐expressing tumor cells (C) and their derived tumor spheres (D) following STAT3 or MYC restoration.

Fig 4: RINES suppresses tumor sphere formation by inhibiting stemness regulators via its RING domain. (A) Heatmap showing expression of RINES and selected stemness regulators. Analysis was performed on analysis of NCI60 cell lines from the CellMiner database using TMM‐FPKM (log2)‐normalized H3K4me3 and H3K27ac ChIP‐seq data. (B) Western blot analysis demonstrating reduced levels of stemness regulators (STAT3, MYC and MDM2) in tumor cell lines expressing RINES, but not isoform 2, under normal culture or 24 h serum starvation. MB231 cells expressing RINES or isoform 2 were cultured in serum‐free medium. (C) Western blot analysis of STAT3, MYC, and MDM2 expression in tumor cell lines expressing RINES or the RING‐domain mutant. (D) MG132 (proteasome inhibitor, 10 µM for 6 h) treatment reversed RINES‐mediated downregulation of endogenous STAT3 and MYC in KYSE510 and HCT116 cells expressing a control vector, RINES, or the RING‐domain mutant, as shown by Western blot. (E) Representative images of colony formation assays using monolayer‐cultured tumor cells expressing RINES or the RING‐domain mutant. Quantitative analysis of colony numbers is presented as mean ± SD from three independent experiments. Colonies containing more than 50 cells in the empty vector‐transfected cells set as 100 for normalization. p‐values were calculated using one‐way ANOVA with Tukey's post hoc tests. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (F) Representative images of tumor spheroids formed by KYSE510 and NPC43 cells expressing RINES or RING‐domain mutant after 7‐day incubation. Graphs show sphere‐forming efficiency and size distribution. Data are represented as mean ± SD of three independent experiments. Statistical analysis was performed using one‐way ANOVA with Tukey's post hoc test, and representative data were shown. ****, p < 0.0001. Scale bar, 50 µm. (G, H) Western blot analysis of stem cell marker expression in parent cells (G) and tumor spheres (H) expressing RINES or the RING‐domain mutant.

Fig 5: RINES depletion enhances stem‐like properties by upregulating STAT3 and MYC. (A) Tumor sphere formation in KYSE410 and MCF7 cells transfected with control siRNA or RINES siRNA. Representative images of tumor spheres were acquired after 7 days of culture. Graphs quantify sphere‐forming efficiency and size distribution. Data are represented as mean ± SD of three independent experiments by one‐way ANOVA with Tukey's post hoc test, and representative data are shown. ****, p < 0.0001. Scale bar, 50 µm. Con, Control. (B) Western blot analysis revealed elevated STAT3 and MYC protein levels upon RINES knockdown in KYSE410, MCF7, and HEK293T cells, using the indicated antibodies. (C) Interaction between RINES and endogenous STAT3 or MYC. Cell lysates from KYSE410, MCF7, and HEK293T cells with endogenous RINES expression were subjected to co‐IP with IgG or target‐specific antibodies, followed by Western blot detection. (D, E) RINES knockdown extends the half‐life of endogenous STAT3 (D) and MYC (E) proteins. CHX chase assays were performed with 10 µg/mL CHX in KYSE410 and MCF7 cells transfected with control or RINES siRNA. Relative protein levels of STAT3 and MYC (normalized to GAPDH) are presented as mean ± SD of three independent experiments. (F) RINES knockdown reduced the ubiquitination of endogenous STAT3 and MYC, as detected by IP‐Western analysis. (G) Schematic model illustrating the mechanism by which RINES regulates cancer stemness. RINES mediates RING domain‐dependent ubiquitination and degradation of the stemness regulators STAT3 and MYC during tumorigenesis.