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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. 6 — a ATF4 expression measured by RT-qPCR in siLacZ- and siFANCD2-transfected cells, treated or not with 1 µM TG (left panel). n = 5 independent experiments. ****p < 0.0001. Western Blot detection of ATF4 in cytoplasmic and nuclear fractions of siLacZ- or siFANCD2-transfected cells treated (+) or not (−) with 1 µM TG (right panel). Vinculin and lamin A/C were used as loading controls for cytoplasmic and nuclear fractions, respectively. b Expression of ATF4 target genes measured by RT-qPCR after control, FANCD2, ATF4, or FANCD2 and ATF4 siRNA transfection. n = 3 independent experiments. *p = 0.0194 (PSAT1_siFANCD2), *p = 0.0134 (PSAT1_siATF4), **p = 0.0015 (PSAT1_siFANCD2 + siATF4), *p = 0.0105 (PSAT1_siFANCD2 + siATF4 vs. PSAT1_siFANCD2); **p = 0.0048 (CHAC1_siFANCD2), **p = 0.015 (CHAC1_siATF4), ****p < 0.0001 (CHAC1_siFANCD2 + siATF4), **p = 0.0022 (CHAC1_siFANCD2 + siATF4 vs. CHAC1_siFANCD2); *p = 0.0239 (PKC2_siFANCD2), *p = 0.0201 (PKC2_siFANCD2 + siATF4 vs. PKC2_siFANCD2). c FHIT mRNA level measured by RT-qPCR in siLacZ- or siFANCD2-transfected cells treated or not with 1 µM TG or 10 µM CCCP and ISRIB (500 nM) for 8 h. n = 7 (siLacZ, siFANCD2, siLacZ + TG, siFANCD2 + TG), n = 5 (siLacZ + CCCP, siFANCD2 + CCCP), n = 4 (siLacZ + ISRIB, siFANCD2 + ISRIB, siLacZ + TG + ISRIB, siFANCD2 + TG + ISRIB, siLacZ + CCCP + ISRIB, siFANCD2 + CCCP + ISRIB) independent experiments. ****p < 0.0001 (siFANCD2), ****p < 0.0001 (siFANCD2 + ISRIB), ***p = 0.0004 (siLacZ + TG), ****p < 0.0001 (siFANCD2 + TG), **p = 0.0047 (siLacZ + TG + ISRIB), *p = 0.0241 (siLacZ + TG + ISRIB vs. siLacZ + TG), ***p = 0.0003 (siFANCD2 + TG + ISRIB), *p = 0.026 (siLacZ + CCCP), ****p < 0.0001 (siFANCD2 + CCCP), *p = 0.0202 (siLacZ + CCCP + ISRIB), ***p = 0.0001 (siFANCD2 + CCCP + ISRIB). d PARK2 mRNA level measured by RT-qPCR in siLacZ-transfected or siFANCD2-transfected cells treated or not with 1 µM TG or 10 µM CCCP and ISRIB (500 nM) for 8 h. n = 10 (siLacZ, siFANCD2), n = 8 (siLacZ + CCCP, siFANCD2 +CCCP), n = 7 (siLacZ + TG, siFANCD2 + TG), n = 4 (siLacZ + ISRIB, siFANCD2 + ISRIB, siLacZ + TG + ISRIB, siFANCD2 + TG + ISRIB, siLacZ + CCCP + ISRIB, siFANCD2 + CCCP + ISRIB) independent experiments. ****p < 0.0001 (siFANCD2), ****p < 0.0001 (siFANCD2 + ISRIB), **p = 0.0045 (siLacZ + TG), ****p < 0.0001 (siFANCD2 + TG), *p = 0.0312 (siLacZ + TG + ISRIB), *p = 0.0328 (siLacZ + TG + ISRIB vs. siLacZ + TG), ****p < 0.0001 (siFANCD2 + TG + ISRIB), *p = 0.022 (siLacZ + CCCP), ****p < 0.0001 (siFANCD2 + CCCP), *p = 0.0203 (siLacZ + CCCP + ISRIB), ****p < 0.0001 (siFANCD2 + CCCP + ISRIB). e CHOP mRNA level measured by RT-qPCR in siLacZ-transfected or siFANCD2-transfected cells treated or not with 1 µM TG or 10 µM CCCP and ISRIB (500 nM) for 8 h. n = 7 (siLacZ, siFANCD2, siLacZ + CCCP, siFANCD2 + CCCP), n = 6 (siLacZ + TG, siFANCD2 + TG), n = 4 (siLacZ + ISRIB, siFANCD2 + ISRIB, siLacZ + TG + ISRIB, siFANCD2 + TG + ISRIB, siLacZ + CCCP + ISRIB, siFANCD2 + CCCP + ISRIB) independent experiments. *p = 0.0221 (siFANCD2 + ISRIB), ***p = 0.0001 (siLacZ + TG), ***p = 0.0001 (siFANCD2 + TG), ***p = 0.0002 (siLacZ + TG + ISRIB), ****p < 0.0001 (siFANCD2 + TG + ISRIB), *p = 0.0382 (siFANCD2 + TG + ISRIB vs. siLacZ + TG), ****p < 0.0001 (siLacZ + CCCP), *p = 0.0477 (siFANCD2 + CCCP), ****p < 0.0001 (siLacZ + CCCP + ISRIB), **p = 0.0046 (siFANCD2 + CCCP vs. siLacZ + CCCP), **p = 0.0033 (siLacZ + CCCP + ISRIB vs. siLacZ + CCCP), **p = 0.0033 (siFANCD2 + CCCP + ISRIB vs. siLacZ + CCCP). f BIP mRNA level measured by RT-qPCR in siLacZ-transfected or siFANCD2-transfected cells treated or not with 1 µM TG or 10 µM CCCP and ISRIB (500 nM) for 8 h. n = 7 (siLacZ, siFANCD2, siLacZ + CCCP, siFANCD2 + CCCP), n = 6 (siLacZ + TG, siFANCD2 + TG), n = 4 (siLacZ + ISRIB, siFANCD2 + ISRIB, siLacZ + TG + ISRIB, siFANCD2 + TG + ISRIB, siLacZ + CCCP + ISRIB, siFANCD2 + CCCP + ISRIB) independent experiments. ****p < 0.0001 (siLacZ + TG), ****p < 0.0001 (siFANCD2 + TG), ****p < 0.0001 (siLacZ + TG + ISRIB), ****p < 0.0001 (siFANCD2 + TG + ISRIB), **p = 0.008 (siLacZ + CCCP), *p = 0.0244 (siFANCD2 + CCCP), *p = 0.0361 (siFANCD2 + TG + ISRIB vs. siLacZ + TG), *p = 0.0467 (siFANCD2 + CCCP + ISRIB vs. siLacZ + CCCP). g Left, graphical representation of mitochondrial UPR activation of CFS genes. Right, expression of large CFS genes measured by RT-qPCR after control, FANCD2, UBL5, or FANCD2 and UBL5 siRNA transfection. n = 4 (siLacZ, siFANCD2, siFANCD2 + siUBL5), n = 3 (siUBL5) independent experiments. **p = 0.014 (FHIT_siFANCD2), **p = 0.0062 (FHIT_siFANCD2 + siUBL5), *p = 0.0461 (FHIT_siFANCD2 + siUBL5 vs. FHIT_siFANCD2); ***p = 0.0003 (IMMP2L_siFANCD2), ****p < 0.0001 (IMMP2L_siUBL5), **p = 0.0062 (IMMP2L_siFANCD2 + siUBL5), **p = 0.0025 (IMMP2L_siFANCD2 + siUBL5 vs. IMMP2L_siFANCD2); **p = 0.0067 (PARK2_siFANCD2), ***p = 0.0004 (PARK2_siUBL5), *p = 0.0328 (PARK2_siFANCD2 + siUBL5 vs. PARK2_siFANCD2). h Chromosome fragility in cells transfected with control, FANCD2, UBL5, or FANCD2 and UBL5 siRNA and treated with 0.3 µM APH, scored as the mean number of breaks per metaphase. A total of 202 (siLacZ), 202 (siFANCD2), 194 (siUBL5), and 202 (siFANCD2 + siUBL5) metaphases were analyzed from n = 4 independent experiments. *p = 0.035 (siFANCD2). Error bars are standard error of the mean (SEM).