Fig 2: Knockdown of CD44v10 isoform inhibits the proliferation of HA−/low binding subpopulation. (A) Cell proliferation was evaluated using a CCK-8 assay after CD44v10 knockdown. (B) Cell proliferation was also assessed using the EdU incorporation assay (magnification, ×100). (C) Proliferative ability of HA−/low cells transfected with CD44v10 was detected by colony formation. *P<0.05, **P<0.01, ***P<0.001 vs. sicontrol group. (D) Viability of MDA-MB-231 and BT-549 HA−/low cells after control siRNA or CD44v10 siRNA transfection and treatment with anti-CD44 mAb or NIgG (10 µg/ml) for 48 h was measured using a CCK-8 assay. Unpaired Student's t-tests were used to analyze the data. Data are presented the mean ± SD of three experiments; n=3. **P<0.01 vs. siCD44v10 + NIgG group. NIgG, normal mouse IgG; CCK, Cell Counting Kit; HA, hyaluronan; siRNA/si, small interfering RNA; CD44v, CD44 variant; EdU, 5-ethynyl-2′-deoxyuridine; mAb, monoclonal antibody.

Fig 3: miR-328-3p worked as a cancer suppressor in STAD via targeting CD44. To further indicate whether miR-328-3p regulated the behaviors of STAD cells in a CD44-dependent manner, AGS cells were cotransfected with miR-328-3p inhibitors and si-CD44. (a) qRT-PCR and Western blot were utilized to detect the transfection efficiency of CD44 knockdown. (b) CCK-8 assay was used to assess the proliferation rate of AGS cells. (c) Western blot assay was used to detect the expression levels of apoptosis-related proteins. (d, e) The migration and invasion of AGS cells were assessed with (d) wound healing and (e) Transwell assays. (f) The expression levels of SOX2, NANOG, EPCAM, CD133, and CD166 were determined by qRT-PCR. Scale bar = 100 μm. All experiments were performed in triplicate. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Fig 4: THOC5 is required for THOC2‐mediated stemness enhancement in radioresistant TNBC cells. A) THOC2 was silenced in MDA‐MB‐231‐RR and 436‐RR cells using a lentiviral system with two different shRNAs. The protein expression of THOC5 was detected by WB. GAPDH was used as the loading control. B) The enrichment of GAPDH, OCT4, NANOG, and SOX2 mRNAs against THOC5 and IgG antibodies in TNBC cell lines was analyzed by RIP assay. THOC5 was silenced in MDA‐MB‐231‐RR and 436‐RR cells using a lentiviral system with two different shRNAs. C) The protein expression of THOC2, THOC5, OCT4, NANOG, and SOX2 was detected by WB. GAPDH was used as the loading control. D) The stemness of TNBC cell lines was evaluated by mammosphere formation assay, and the formation efficiency was calculated. Representative images are shown at 100 × magnification. E) The percentage of CD44+CD24−/low cells in MDA‐MB‐231‐RR cells was detected using flow cytometry. F) The percentage of ALDH+ cells in MDA‐MB‐436‐RR cells was detected using flow cytometry. G) The total mRNA expression of NANOG and SOX2 in TNBC cell lines was detected by qRT‐PCR. H) The relative expression of polysome‐associated NANOG and SOX2 mRNAs (fractions 7–12) was detected by qRT‐PCR. I) The intracellular distribution of NANOG and SOX2 mRNAs was evaluated by RNA‐FISH. Representative images are shown at 630 × magnification. Red represents target mRNA, while blue represents the nuclei. J) The enrichment of NANOG and SOX2 mRNAs against THOC2 and IgG antibodies in TNBC cell lines was analyzed by RIP assay. *P < 0.05, **P < 0.01, and ***P < 0.001 versus parental cells or sh‐control group (n = 3).

Fig 5: ING4 promoted stemness enrichment of RCC cells through the activation of the p38 MAPK/type I IFN-stimulated gene signaling pathway. (A–B) Western blot showed levels of p38 MAPK and Erk1/2 activation in ING4-overexpressed (ING4) or knockdown (sgRNA-ING4) Ketr-3 and 786-O cells, and the relative protein expression of p-p38 or p-Erk1/2 was normalized to that of the respective p38 or Erk1/2. (C–D) Real-time PCR analyzed the mRNA expression levels of ISGs including OAS2, MX2, IFITM1, and IFITM2 in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 treatment for 24 h. (E–F) Photographs and number of cell spheres in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 pretreatment for 24 h (n = 3). (G–H) Western blot tested the protein expression levels of stem cell markers OCT4, CD44, MYC, and NANOG in 786-O and Ketr-3 cells with ING4 overexpression (ING4) or vector control (Vec) after 0.1%DSMO or 5 μM SB203580 treatment for 24 h and relative protein expression was normalized to that of the respective α-tubulin. Note: images magnification, ×100; Scale bar, 100 μm; Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.001, ns: no significance.