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. 2021 Oct 13;40:319. doi: 10.1186/s13046-021-02117-z

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Expression and genetic manipulation of L1CAM in OCSC. (A) qRT-PCR for L1CAM and the stemness-related genes NANOG, POU5F1 and NT5E on second-generation spheres from OC primary cells. Data are expressed as relative mRNA level (2^-ΔΔCt) and normalized to the corresponding adherent cultures (dashed line). Data refer to two independent primary OC samples (OC1 and OC2). (B) Immunoblotting on L1CAM-silenced OVCAR3 cells. L1CAM silencing was efficiently achieved through two different shRNAs. Vinculin served as loading control. The panel shows a single blot, intervening lanes were removed for clarity reasons. (C) Sphere forming efficiency (SFE) on L1CAM-knockdown and control OVCAR3 cells. (D) Sphere formation assay conducted on L1CAM-knockdown or control OVCAR3 cells upon rescuing L1CAM expression. (E) Immunoblot for L1CAM in L1CAM- and mock-transduced Ov90 cells. ERK1/2 served as loading control. The panel shows a single blot, intervening lanes were removed for clarity reasons. (F) L1CAM-trasduced Ov90cells were subjected to sphere formation assay for three consecutive sphere generations. (G) Sphere formation assay conducted in medium depleted of both EGF and FGF2. (H) Three different primary ovarian cancer samples (OC3, OC4 and OC5) were sorted according to L1CAM expression (see Supplementary Fig. S4) and subjected to sphere formation assay. For each analysis, data are expressed as means ± SEM from three independent experiments. Comparisons between experimental groups were done with two-sided Student’s t-tests; *p < 0.05, **p < 0.01, ***p < 0.001