Fig 1: HSF1 is critical for maintenance of LSC self-renewal.a Engraftment of three different primary AML LSCs in the presence or absence of CRISPR-mediated HSF1KD (started 4 mice/AML). *p = 0.0404, **p = 0.0003, ***p = 0.0006. b ATP production in control or HSF1 knockdown AML cells (post-transplantation from a) measured by Seahorse assay (n = 3 independent replicates). AML-a: *p = 0.0046. **p = 0.0349; AML-b: *p = 0.0201. **p = 0.0124; AML-c: *p = 0.0325. **p = 0.0033; c The expression of HSP90, SDHC, and HSF1 proteins in SISU-102 treated mouse MLL-AF9 cells and the human AML cell lines MV4–11 and NOLM-1 (n = 3 independent replicates). ACTIN is used as a load control. d Cell growth of murine MLL-AF9 cells (*p = 1.6 × 10-5, **p = 3.1 × 10-5, ***p = 6.1 × 10-5) and human AML cell line MV4–11 (*p = 4.8 × 10-3, **p = 2.3 × 10-4, ***p = 1.6 × 10-4) and NOLM-1 (*p = 6.3 × 10-4, **p = 3.01 × 10-5, ***p = 6.38 × 10-5) in the presence of absence of SISU-102 (n = 3 independent replicates). e OCR in Hsf1fl/flcreER LSCs (*p = 8.87 × 10-31, **p = 1.09 × 10-7) or human AML cell line MV4–11 (*p = 4.89 × 10-13, **p = 4.97 × 10-12) and NOLM-1 (*p = 1.19 × 10-18, **p = 0.00028) treated with or without SISU-102 (n = 8 independent replicates). f Engraftment of human AML cell line MV4–11 (n = 5 mice/group, *p = 9.8 × 10-5) or three different primary AML cells in the presence or absence of SISU-102. AML-d: *p = 1.8 × 10-5, n = 4 mice/group, and AML-e: *p = 8.98 × 10-6, Con n = 4 mice, SISU-102, n = 5 mice; AML-f: *p = 0.0011, Con n = 5 mice, SISU-102, n = 4 mice. g Engraftment of human BM CD34+ HSPCs treated with (n = 4 mice) or without (n = 3 mice) SISU-102 (5 mg/kg, IP, daily). In (a, b, d–f), two-tailed t test was used, data are presented as mean values ± SEM. Source data are provided as a Source Data file.
Fig 2: HSF1 regulated genes are involved in multifaceted LSC functions.a–c RNA-seq analysis of sorted Contol (Con) or Hsf1KO MLL-AF9 LSCs (isolated from 2 full blown individual leukemia mice for each genotype). Hsf1 ablation (a) downregulated its known targets—heat shock protein genes; (b) upregulated genes related to myeloid differentiation (Itgam, Elane, Mpo) and downregulated stemness related genes (CD34, Flt3, Bcat1), and (c) genes related to OXPHOS. d–f GSEA revealed that Hsf1 deletion impaired stem cell signature (d), down-regulated genes related to OXPHOS (e) and TCA cycle (f). g CUT&RUN peak call enrichment analyses. KEGG and Gene ontology terms derived from genes involved in top 8000 peaks. h CUT&RUN demonstrated HSF1 binds Sdhc directly. H3K4me3 shows that HSF1 binding occurs in active chromatin region. Binding to genes encoding heat shock proteins (Hspd1 and Hspe1) and IgG binding were used as positive and negative controls, respectively.
Fig 3: HSF1 is a key regulator of oxidative phosphorylation in AML.For experiments (a–m), Hsf1fl/flcreER mouse MLL-AF9 LSPCs were treated with or without (Con) 200 nM 4-OHT for different times. a 2-NBDG uptake (n = 3 independent replicates). b Mitochondrial membrane potential (Mitotracker, n = 3 independent replicates). c Mitochondrial superoxide (Mitosox, n = 4 independent replicates), *p = 0.0017. d, e Mitochondrial ROS levels measured by H2-DCFDA (d, n = 4 independent replicates, *p = 0.0106) or CellROX (e, n = 3 independent replicates, *p = 0.0016). f, g OCR (f, *p = 5.9 × 10-18, **p = 4.3 × 10-11) and ECAR (g, *p = 5.3 × 10-7, **p = 8.8 × 10-8) measured by Seahorse (n = 8, independent replicates each). h ATP production (n = 3 independent replicates). *p = 3.57 × 10-5, **p = 0.000795. i Leukemia cell intermediate metabolites (n = 5 independent replicates) using high-resolution Gas chromatograph mass spectrometry (GC/MS). j 13C6-Glucose tracing (measuring leukemia cell intermediate metabolites) using GC/MS. Left, illustration of glucose metabolism. White circles, 12C carbons; blue circles, 13C carbons. M + 2 and M + 3 refer to the number of 13C carbons.; Right, TCA intermediates: citrate (*p = 0.00167), succinate (*p = 0.0121), fumarate, and malate (*p = 0.0011) and intracellular metabolites: pyruvate, alanine, lactate (*p = 0.0146), glutamate (*p = 0.00014), and aspartate (*p = 0.00098). Data are the isotopologue distribution relative to abundance and normalized to the control group, plotted as mean values and individual data points from n = 5 cultures. k Expression of key electron transport chain complex (ETC) I, II, III, and IV components by Western blotting (n = 3 independent replicates). l qPCR analysis of expression of Sdha (*p = 0.0008) and Sdhc (*p = 8.2 × 10-7) (n = 3 independent replicates). m ETC complex II activity (*p = 0.00575) (n = 3 independent replicates). n Expression of exogenous SDHC protein in Hsf1fl/flcreER LSPCs (n = 2 independent replicates). o, p Leukemia cell proliferation (o, *p = 0.00078) and colony formation (p, *p = 0.00024) in empty vector or SDHC transduced Hsf1fl/flcreER LSCs in the presence or absence of 200 nM of 4-OHT (n = 3 independent replicates, each). q OCR in empty vector or SDHC transduced Hsf1fl/flcreER LSPCs treated with or without 200 nM 4-OHT (n = 7 independent replicates). *p = 0.00066, **p = 2.2 × 10-10, ***p = 4.86 × 10-6. r ETC complex II activity in empty vector or SDHC transduced Hsf1fl/flcreER leukemia cells treated with or without (Con) 200 nM 4-OHT (n = 4 independent replicates). *p = 0.00056, **p = 0.0044. In (c–e, h, j, l–m, r), two-tailed t test was used, data are presented as mean values ± SEM. s Survival curve of sublethally irradiated recipient mice receiving vector or SDHC transduced Hsf1fl/flcreER LSCs treated with vehicle (n = 5 mice/group) or TAM (n = 9 mice/group). *p = 0.0001. Source data are provided as a Source Data file.
Fig 4: NRF2 negatively regulates the expression of FOCAD. To downregulate the expression of NRF2, A549 cells were treated with brusatol (10 nM, 30 nM, 50 n M for 16 h) (A–B) or siRNA (10 nM for 48 h) (C–D). NRF2 knockout (KO) cell line was also established using CRISPR/Cas9 (C–D). Then, the cell samples were harvested for western blot (A and C) and qPCR detection (B and D). In addition, biotinylated DNA probes (41 bp) spanned the ARE containing sequences in the promoter regions of human FOCAD gene, and the DNA-protein complexes were pulled down using streptavidin beads for immunoblot detection (E). To confirm the model, RPA1 knockout cell line (A549-RPA1−/−) was also established and pGL3-FOCAD-ARE plasmid and hRluc/TK plasmid were cotransfected into the cell using Lipofectamine 3000. 24 h later, the cells were treated with brusatol (50 nM for 16 h) or ML385 (5 μM for 16 h) for luciferase activity assay (F). Results were expressed as mean ± SD (n = 3), and the P value less than 0.05 was considered statistically significant. *: P < 0.05 compared with the control (without any treatment) group.
Fig 5: KD of ACC1 in U251 cells elevates DNMT1 expression via P300. (A) RT-qPCR of P300 mRNA after siP300 transfection in wild-type U251 cells. (B) RT-qPCR of P300 mRNA after siP300 transfection in ACC1 KD cells. (C) RT-qPCR of DNMT1 mRNA after siP300 transfection. (D) RT-qPCR of SDHA, SDHB, SDHC and SDHD mRNA after siP300 transfection. (E) Methylation-specific PCR of SDHB promoter methylation after siP300 transfection. (F) Semi-quantification of SDHB promoter methylation levels after siP300 transfection. (G) Flow cytometric analysis of ROS after siP300 transfection. (H) Quantification of cellular ROS fluorescence signals after siP300 transfection. (I) Images of Transwell migration/invasion assays after siP300 transfection. (J) Quantification of Transwell migration/invasion assays after siP300 transfection. (K) Wound-healing images after siP300 transfection. (L) Wound-healing assay quantification after siP300 transfection. (M) WB of ACC1, SDHA, SDHB, H3K9ac, P300, DNMT1, vimentin, H3, fibronectin and PAI-1 after siP300 transfection. (N) Semi-quantification of ACC1, SDHA, SDHB, H3K9ac/H3, P300, DNMT1, vimentin, fibronectin and PAI-1 protein levels after siP300 transfection. Scale bars, 100 μm. Error bars represent the mean ± standard deviation from three independent experiments. *P<0.05; **P<0.01; ***P<0.001. #P<0.05; ##P<0.01; ###P<0.001. ACC1, acetyl-CoA carboxylase 1; DNMT, DNA methyltransferase; SDH, succinate dehydrogenase; si, small interfering; U, unmethylated; M, methylated; ROS, reactive oxygen species; H3K9ac, histone H3 acetylation at lysine 9; PAI-1, plasminogen activator inhibitor-1; WB, western blotting; KD, knockdown; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; NC, negative control.
Supplier Page from Abcam for Complex II Activity Assay Kit (Colorimetric)