Fig. 7: NFIC KD reduces growth, survival and clonogenicity of MLL::AF9 pre-LSCs and induces differentiation and apoptosis.

a Expression of Nfic mRNA in pre-LSCs clones isolated from MLL::AF9 transformed mice (n = 5) as compared to their normal counter-part granulo-monocytic myeloid progenitor (GMP) cells (n = 4). Each dot represents an individual mouse. Data was analyzed using Mann-Whitney test. b Line graph represents growth curve of MLL::AF9 (with GFP) transduced pre-LSCs after Nfic knockdown. MLL::AF9 transformed pre-LSCs clones (Clone 1 and 2) form two different mice models were repeat-infected with either Nfic shRNAs (sh539 and sh545) or scrambled (shScr) vectors containing m-Cherry. Cells were seeded 48 h post infection. Total cell counts were performed every 24 h for 4 days. Cells were initially gated on GFP for MLL::AF9 and then on m-Cherry. 7-AAD was used to exclude dead cells. Data are mean ± 1 SD with *p < 0.05 and **p < 0.01, analyzed by Dunnett’s multiple comparison test as compared to scramble control. c Colony formation assay. The bar graph represents number of colonies per 2000 cells seeded. MLL::AF9 transformed cells were infected with Nfic shRNAs or scrambled for 48 h, sorted as GFP⁺/m-Cherry⁺ and seeded in six-well plates containing MethoCult for primary, secondary, and tertiary colony. Colonies were counted and phenotyped after seven days and re-seeded for subsequent colony. Data represented are mean±1 SD (n = 4) with *p < 0.05, **p < 0.01 and ***p < 0.001 as analyzed by Tukey’s multiple comparison test. d Bar graph represents Annexin-V⁺ cells. MLL::AF9 transformed pre-LSCs were infected with Nfic shRNAs or scrambled for 72 h and analyzed for apoptosis assay. 7-AAD was used for necrotic and late-apoptotic cells. Each bar graph shows sum of both Annexin-V⁺ (early apoptotic) and 7-AAD⁺/Annexin-V⁺ (late apoptotic) cell populations. Data represented are mean ± 1 SD (n = 4) with *p < 0.05 and **p < 0.01 as analyzed by Dunnett’s multiple comparison test. e Flow cytometric histogram plots for c-kit, Mac and Gr-1 expression in tertiary colonies of MLL::AF9 transformed pre-LSCs infected with Nfic shRNAs as compared to control (n = 4).