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. 2021 Jan 29;4:127. doi: 10.1038/s42003-021-01647-8

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© The Author(s) 2021

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Left, RT-qPCR-based analysis of CFS gene expression in siLacZ- and siFANCD2-transfected cells treated or not with 10 µM CCCP for 8 h. Right, RT-qPCR-based analysis of CFS gene expression in siLacZ- and siFANCD2-transfected cells treated or not with 1 µM TG for 8 h. n = 6 (FHIT_siLacZ), n = 4 (FHIT_siFANCD2), n = 6 (FHIT_siLacZ_CCCP), n = 4 (FHIT_siFANCD2_CCCP), n = 6 (PARK2_siLacZ), n = 4 (PARK2_siFANCD2), n = 6 (PARK2_siLacZ_CCCP), n = 4 (PARK2_siFANCD2_CCCP), n = 3 (IMMP2L) independent experiments for cells treated or not with CCCP; and n = 5 (FHIT), n = 4 (PARK2), n = 3 (IMMP2L) for cells treated or not with TG. ****p < 0.0001 (FHIT_siFANCD2), *p = 0.0207 (FHIT_siLacZ_CCCP), ***p = 0.0007 (FHIT_siFANCD2_CCCP), *p = 0.0174 (FHIT_siFANCD2_CCCP vs. FHIT_siLacZ_CCCP), **p = 0.0048 (PARK2_siFANCD2), *p = 0.047 (PARK2_siLacZ_CCCP), ***p = 0.0008 (PARK2_siFANCD2_CCCP), *p = 0.0104 (IMMP2L_siFANCD2), **p = 0.0037 (IMMP2L_siFANCD2_CCCP), *p = 0.023 (IMMP2L_siFANCD2_CCCP vs. IMMP2L_siLacZ_CCCP); ****p < 0.0001 (FHIT_siFANCD2), *p = 0.0038 (FHIT_siLacZ_TG), ****p < 0.0001 (FHIT_siFANCD2_TG), ***p = 0.0003 (FHIT_siFANCD2_TG vs. FHIT_siLacZ_TG), **p = 0.003 (PARK2_siFANCD2), **p = 0.0058 (PARK2_siLacZ_TG), ***p = 0.0002 (PARK2_siFANCD2_TG), ***p = 0.0006 (PARK2_siFANCD2_TG vs. PARK2_siLacZ_TG), **p = 0.008 (IMMP2L_siFANCD2), ***p = 0.0006 (IMMP2L_siLacZ_TG), **p = 0.0052 (IMMP2L_siFANCD2_TG), **p = 0.0087 (IMMP2L_siFANCD2_TG vs. IMMP2L_siLacZ_TG). b Left, examples of immunofluorescence staining of FANCD2 in cells treated with TG or CCCP or in cells that were not treated (NT); FANCD2 is shown in red, and DNA (DAPI) is in blue in the merged image. Right, percentage of FANCD2-positive nuclei; FANCD2 foci were counted in a total of 335 (NT), 318 (TG), and 330 (CCCP) nuclei from n = 3 independent experiments, and nuclei with more than five spots were quantified. Scale bars, 5 μM. c Immunofluorescence staining of FANCD2 in metaphase cells treated or NT with TG or APH. Scale bars, 5 μM. d FANCD2 ChIP followed by qPCR in cells treated or not with 1 µM TG or 10 µM CCCP for 8 h. The results are expressed as the percentage of the input. Chromosome 7 (Chr 7), Chromosome 16 (Chr 16), and Chromosome 3 (Chr 3) were used as control regions close to the IMMP2L, WWOX, and FHIT genes, respectively. n = 3 independent experiments. **p = 0.0084 (IMMP2L_NT), **p = 0.002 (IMMP2L_TG), ***p = 0.001 (IMMP2L_CCCP), **p = 0.002 (WWOX_NT), **p = 0.0004 (WWOX_TG), *p = 0.139 (WWOX_CCCP), ****p < 0.0001 (FHIT_NT), ****p < 0.0001 (FHIT_TG), *p = 0.161 (FHIT_CCCP). e Expression of large CFS genes measured by RT-qPCR after control, FANCD2, SPG7, or FANCD2 and SPG7 siRNA transfection. n = 3 independent experiments. ****p < 0.0001 (FHIT_siFANCD2), ****p < 0.0001 (FHIT_siSPG7), ***p = 0.0004 (FHIT_siFANCD2 + siSPG7), **p = 0.0036 (PARK2_siFANCD2), **p = 0.0066 (PARK2_siSPG7), **p = 0.0014 (PARK2_siFANCD2 + siSPG7), ****p = 0.0001 (IMMP2L_siFANCD2), ***p = 0.0006 (IMMP2L_siSPG7), ***p = 0.0005 (IMMP2L_siFANCD2 + siSPG7). Error bars are standard error of the mean (SEM).