Fig 2: Solute carrier family 34 member 2 (SLC34A2) promotes esophageal squamous cell carcinoma (ESCC) cell autophagy and proliferation by upregulating STX17. (a) Immunoprecipitation (IP) combined with Coomassie brilliant blue staining and mass spectrometry was used to identify the interacting proteins of SLC34A2. (b) The interaction between three top‐ranking autophagy‐related proteins and SLC34A2 was tested by IP and western blot (WB) in 293 T cells transfected with SLC34A2‐HA or vector‐HA. (c) Co‐IP was used to verify the SLC34A2‐STX17 interaction in ESCC cells. (d) The STX17 protein level was examined in SLC34A2‐ overexpressed/−silenced cells by WB. (e) WB was utilized to examine the levels of LC3B and P62 in SLC34A2‐overexpressed ESCC cells under starvation conditions after silencing STX17. (f) The mRFP‐GFPLC3B reporter was performed to monitor the autophagic flux of SLC34A2‐overexpressed ESCC cells with transient STX17 knockdown. Scale bar, 5 μm. (g, h) Cell counting kit‐8 (CCK‐8) and 5‐ethynyl‐2′‐deoxyuridine (EdU) assays were used to detect the proliferation ability of SLC34A2‐overexpressed cells with transient STX17 knockdown. Scale bar, 20 μm. (i) Five nude mice in each group were subcutaneously injected with KYSE520‐SLC34A2 cells with STX17 knockdown, KYSE520‐SLC34A2 cells and KYSE520 wild‐type cells. The mean tumor volumes (cm3) and weights (g) were measured 3 weeks later. The grayscale value of the target protein in the WB was quantified using ImageJ software and normalized to α‐tubulin. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: SLC34A2 directly binds to the promoter of MIR25 and increases its expression in neuroblastoma cells and spheroids. (A) Diagram of MIR25 promoter containing SLC34A2 binding site and corresponding mutant site. (B) MiR‐25 level was examined in SH‐SY5Y cells and spheroids. (C) MiR‐25 level was determined in SH‐SY5Y cells with SLC34A2 overexpression or spheroids with SLC34A2 knockdown. (D) The luciferase activity of pGL3‐miR‐25 and pGL3‐miR‐25‐mut was measured in SH‐SY5Y cells with or without SLC34A2 overexpression. (E) The luciferase activity of pGL3‐miR‐25 and pGL3‐miR‐25‐mut was measured in SH‐SY5Y cells with or without SLC34A2 knockdown. (F) ChIP assay was performed in SH‐SY5Y cells with anti‐SLC34A or IgG, followed by determination of miR‐25 level. The difference was assayed using one‐way ANOVA with the Tukey–Kramer post hoc test. Data are presented as mean ± SD; n ≥ 3, **P < 0.01 vs control.

Fig 4: SLC34A2 promotes the stemness of neuroblastoma cells. (A) Representative image of spheroids formed by SH‐SY5Y cells. Scale bar, 100 μm. (B,C) SLC34A2 expression was detected in SH‐SY5Y cells and spheroids. (D,E) Expression of stemness markers (ALDH1 and Nanog) was examined in SH‐SY5Y cells and spheroids. (F,G) Expression of stemness markers was determined in SH‐SY5Y cells with or without SLC34A2 overexpression. (H) The capacity of spheroid formation was evaluated in cells depicted in (F). Scale bar, 100 μm. (I,J) Expression of stemness markers was measured in SH‐SY5Y cell spheroids with or without SLC34A2 knockdown. (K) The capacity of spheroid formation was detected in spheroids described in (I). (L,M) The overexpression and knockdown efficiency of SLC34A2 was confirmed in SH‐SY5Y cells and spheroids, respectively. The difference was assayed using one‐way ANOVA with the Tukey–Kramer post hoc test. Data are presented as mean ± SD; n ≥ 3, **P < 0.01 vs control.

Fig 5: Overexpression of solute carrier family 34 member 2 (SLC34A2) promotes esophageal squamous cell carcinoma (ESCC) cell proliferation in vitro and in vivo. (a, b) Western blot (WB) and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) were performed to analyze the SLC34A2 protein and mRNA levels in stably SLC34A2‐ overexpressed KYSE520 and KYSE140 cells. (c–h) Cell counting kit‐8 (CCK‐8) (c), colony formation (d) and 5‐ethynyl‐2′‐deoxyuridine (EdU) (e–h) assays were conducted to measure the proliferative capacity of SLC34A2‐overexpressed ESCC cells. Scale bar, 20 μm. (i, j) Cell cycle distribution in SLC34A2‐overexpressed ESCC cell was assessed by flow cytometry. (k) The xenograft tumor photographs of nude mice injected with KYSE520/SLC34A2 cells or KYSE520/control cells (n = 5). The tumor volume (mm3) and weight (g) were measured. HE and Ki‐67 staining of xenograft tumors were evaluated (magnification, ×400). The grayscale value of the target protein in the WB was quantified using ImageJ software and normalized to GAPDH. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.