Fig 2: Lowering ATF3 improves the effect of 5-FU on CRC MCTS.A Plot for protein interactions among genes ATF3, ATF4, DDIT4, TRIB3 and FGD6 by the STRING interaction network. B Expression levels of ATF3, ATF4, DDIT4 and TRIB3 in HCT116, DLD-1 and HT-29 cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. C Expression levels of ATF3, DDIT4 and TRIB3 were assessed in COAD tissues and normal adjacent tissues using RNA-Seq data from TCGA (* p < 0.05). Cohort size: (num)normal adjacent tissues = 349; (num) COAD tissues = 275. D Western blot analysis of the co-IP complex from cells treated with DMSO or 100 nM sangivamycin for 24 h with ATF3 antibody immobilized on Dynabeads®. Tubulin is the loading control for the input conditions. E Expression levels of ATF3, ATF4, DDIT4 and TRIB3 in HCT116 ATF3parental and ATF3KO cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. F Illustration of the interaction between ATF3, ATF4, DDIT4 and TRIB3 based on our results and findings from other groups [79–81]. G ATF3 expression levels in HCT116 and HT-29 cells treated with the indicated concentrations of rapamycin for 72 h under monolayer conditions. Actin is the loading control. H ATF3 expression levels in HCT116 MCTS cells treated with rapamycin (10 µM) or Torin-1 (0.5 µM) for 72 h. Actin is the loading control. I Dose response of HCT116 and HT29 MCTS to 5-FU. The data is expressed as the mean ± SD (n = 3). The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. J Combination of rapamycin (10 µM) or Troin1(0.5 µM) with 5-FU under HCT116 MCTS conditions for 72 h. The data is expressed as the mean ± SD (n = 3, unpaired t-test, two-stage step-up (Benjamini, Krieger, and Yekutieli); * p < 0.05, *** p < 0.001). K Combination of rapamycin (10 µM) or Troin1(0.5 µM) with 5-FU under HT29 MCTS conditions for 72 h. CDI = AB/(A × B) where: AB = cell viability value for the combination of rapamycin/torin-1 and 5-FU. A and B = cell viability value for the single treatment rapamycin/torin-1 or 5-FU. A CDI value <1, =1 or >1 indicates that the drugs are synergistic, additive, or antagonistic, respectively. A CDI value less than 0.7 indicates a significant synergetic effect [31].

Fig 3: PP2A-dependent activation of ATF4 and CHOP expression is transcriptionally regulated by TFE3.A mRNA analysis of OV81 and FT246 cell lines assessing transcriptional expression and repression of ATF4, CHOP, ATF3, GADD34, and TRIB3 genes after DT-061 treatment with or without actinomycin D (ActD) pre-incubation, a potent transcription inhibitor that intercalates with the DNA to prevent RNA Polymerase binding. B Western blotting analysis evaluating the protein expression dependency on the transcriptional regulation induced by DT-061 in the OV81 cell line. C Western blotting analysis evaluating cytoplasmic versus nuclear localization and protein expression of TFEB/3, ATF4, and its downstream target genes after 6 h of DT-061 or Tg treatment in OV81. D Western blotting analysis evaluating the effects of DT-061 on the protein expression of TFE3, TFEB, and ISR downstream targets in lung (H358), colorectal (SW620), breast (MDA-MB-231), and prostate (LnCaP and PC3) cell lines. E Protein lysates from experiments represented in Fig. 2C were reanalyzed to evaluate TFEB/3 dephosphorylation status and overall expression under PERKi, DT-061, and combination conditions. Western blotting analysis helped evaluate TFEB/3’s dependency on the UPR-dependent regulation via Calcineurin, in the presence of DT-061. F Protein lysates from Fig. 2F evaluating TFEB/3 expression and dephosphorylation status upon DT-061 treatment, and thus evaluate dependency profiles of DT-061-mediated TFEB/3 effects on PP2A, the UPR-independent mechanism. G Western blotting analysis in C4.2 HGSC cell line evaluating nuclear versus cytoplasmic localization and protein expression of TFEB/3, ATF4, and CHOP after 6 h of Tg, DT-061, calyculin A, or DT + calyculin A treatments. H Representative histology pictures with immunohistochemistry staining (brown) of phospho and total TFE3 for DMA or DT-061 treated tumors from previously published OV81 PDX efficacy studies [21].

Fig 4: PP2A-mediated TFE3, ATF4, and CHOP activation is required for the DT-061-mediated cytotoxic effects.A siRNA experiments knocking down ATF4, TFEB, and TFE3 alone or in dual combination (i.e., ATF4 + TFEB, ATF4 + TFE3, and TFEB + TFE3) were performed in OV81 and FT246 to assess the dependency of DT-061-mediated cytotoxic effects on each of these proteins independently or in combination, evaluating recovery profiles in cell viability by cell titer glo analysis. Graphic representation was generated by calculating %change viability relative to siControl DT-061 treated condition for each respective cell line, after normalization to DMSO control. Data are presented as the mean ± SD (n = 3), (one-way ANOVA with multiple comparisons, comparing the mean of each column with the mean of siControl DT-061 treated column, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). B–E siRNA conditions utilized in A were harvested for B protein and E RNA analysis. B Western blotting assessing the effect of each siRNA alone or in dual combination on the molecular changes of TFEB/3, ISR genes (ATF4, ATF3, CHOP, GADD34, and TRIB3), cell death (cleaved PARP, cleaved Caspase 3, and DR5) and autophagy (LC3) markers. Correlation analysis graph and R2 values comparing the expression of ATF4 with (C) % change viability or (D) each one of its downstream targets (CHOP, TRIB3, and ATF3) in OV81 after DT-061 treatment under RNAi conditions observed in (A, B). E qPCR analysis evaluating ATF4 and its downstream targets’ genetic expression in OV81 is represented as a bar graph. Data presented as the mean ± SD (n = 3) (one-way ANOVA with multiple comparisons, comparing the mean of each column with the mean of siControl DT-061 treated column for each respective target and cell line, ns > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001, top-relative to siControl DMSO, bottom-relative to its own si DMSO). F siCHOP cellular viability experiments in OV81 were pursued to assess dependency profiles of DT-061-mediated cytotoxic effects on CHOP protein expression and evaluate recovery in viability measured by cell titer glo analysis. Graph was generated by calculating %change viability relative to siControl DT-061 condition, after normalization to DMSO. Data presented as the mean ± SD (n = 3), (one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). G Western blotting analysis in OV81 assessing the effect of siCHOP on the molecular signature downstream of ISR genes (ATF4, ATF3, CHOP, GADD34, and TRIB3), and H cell death (cleaved PARP, cleaved Caspase 3, and DR5) and autophagy (LC3) markers after treatment with DT-061 (left). Representation by heatmap of Z-score values obtained after western blot quantification using image J and normalization to Vinculin as the loading control (right). Calculated z-scores were obtained from protein quantification of three independent biological replicates.

Fig 5: FTO influenced proliferation and migration by affecting the expression of TRIB3.A-D Overexpression of TRIB3 rescued the proliferative activity of HaCaT and NHEK cells with FTO knockout, as reflected by CCK-8 and colony formation assays. Semi-quantitative analysis of colony formation assays is shown (n = 3). E, F The Edu incorporation assays revealed that FTO significantly inhibits proliferation of keratinocytes, which can be rescued by overexpression of TRIB3. G, H Overexpression of TRIB3 during FTO knockdown partially rescued the migration of keratinocytes. Images were captured at 0 h and 12 h after the scratch. Semi-quantitative analysis of wound-healing assays is shown (n = 3). I Timeline of in vivo experiments for STZ injections, sgRNA and vector injections, and observation of wound healing models in mice. J Measurements of blood glucose levels in STZ-treated mice. K-M Representative images of cutaneous wounds of diabetic mice, diabetic mice with koFTO injections, diabetic mice with oeTRIB3 injections, and diabetic mice with koFTO and oeTRIB3 injections on days 0, 4, 8, and 12 after wound generation by surgical excision. Rates of wound closure were quantified using ImageJ software and expressed as the percentage of closed wound area (n = 5 per group). The data are presented as the mean ± SD or mean ± SEM (A, B, D, F, H, J, M), and P-values of all data by a two-tailed unpaired t-test are indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.