Fig. 4: NIF Inhibit the STAT3/Fanconi anemia axis.

A Detection of FANCD2, FANCI, and γH2AX by Western blot in SUM159 cell protein extracts after treatment with melphalan, M2, or the combination. Numbers under the blots indicate the fold induction relative to untreated samples. L/S indicates the ratio of monoubiquitinated (L, upper band) to non-monoubiquitinated (S, lower band) FANCD2 or FANCI protein. B Schematic representation of ICLick assay. DNA lesions generated by click-melphalan are detected in red, and its repair can be monitored via the disappearance of the click-labeling. Created in BioRender.com. https://BioRender.com/b62d917. C Quantification of click-labeling (fluorescence intensity) in shCTRL, shCTRL+M2, and shFANCD2 SUM159 cells treated with click-melphalan after 24 h and 72 h of treatment. D Expression of FANCI following the individual treatment of 875 compounds and normalized with DMSO-treated condition used as control. This dot plot has been generated with the DeepCoverMOA web interface. Two families of compounds that significantly decrease FANCI expression are highlighted with MDM2/P53 inhibitors in green or JAK/STAT pathway inhibitors in orange. E CUT&RUN-qPCR of STAT3 in SUM159-KRAB cell under indicated treatment. Data are represented by the fold-change of DNA level normalized on sgSTAT3 condition. P1 and P2 correspond to the two sets of primer designed on STAT3 and FANCI promoter sequence. n = 6 independent experiments. F Schematic representation of FANCI reporter (top panel). Relative luciferase activity measured in SUM159 cells expressing the FANCI promoter after indicated treatment. G mRNA levels of FA genes, measured by qRT-PCR, in SUM159 treated with M2, IL-6, or in combination, normalized with untreated conditions (CTRL). H SUM159 and S68 shCTRL, shFANCD2#1, and shSTAT3 cells exposed to various concentrations of melphalan were subjected to clonogenic survival assays. I Quantification of fluorescence intensity in sgCTRL and sgSTAT3 SUM159-KRAB treated with click-melphalan and measured after 24 h and 72 h of treatment. In (C, E–I), data are shown as mean ± SD. ns (not significant), *P < 0.05, **P < 0.01, and ***P < 0.001 according to one-sided Fisher test (C, E, F, G, I), two-way ANOVA followed by Sidak multiple range test (H). n = 3 independent experiments. Source data are provided as a Source Data file.