Sample quality control and RNA-seq outcome.

Sample quality control and RNA-seq outcome.</p

AOM/DSS-treatment effects.

A. Comparative weight loss as surrogate marker for morbidity. Hltf-deleted designated null -/- (n = 23) and control +/+ (n = 17) mice were weighed on the first (T0) and last weeks (T13) and at 7-day intervals during the treatment protocol. One-way ANOVA (p Hltf-deleted mice at treatment week 6 (arrow). B. Kaplan-Meier survival plot. Comparison of the cumulative survival curve of male Hltf-deleted (n = 109) and control (n = 47) mice indicated differential survival times for Hltf-deleted vs control AOM/DSS-treated mice. C. Blood glucose levels in non-fasting adult male mice. Blood glucose levels were normal for untreated Hltf-deleted vs control, and elevated to the same extent in both groups by AOM/DSS-treatment. In panels A and C, values are mean±SEM. Values with the same letter designation in panel C are not significantly different.</p

Representative histology of AOM/DSS-induced invasive adenocarcinoma.

H&E stained sections from Swiss-rolled colons in wild type control (A; 25x magnification) and Hltf-deleted (B-F) tumors. B. A very large tumor nearly encircles the anal canal (25x magnification). C. Crohn’s disease-like reaction consisting of discrete lymphoid aggregates with germinal centers and invasion at arrow at 25x magnification and 100x magnification (inset). D. Anal canal with invasive tumor (100x magnification). E. Heterotopic (or ectopic) pancreas at arrows (25x magnification). F. Tumor necrosis (arrow) in the anal canal (100x magnification).</p

<i>Hltf</i> regulates OXPHOS.

A. Volcano plot. Differential expression (DE) of ATP5e, Cox7c, Uqcr11, Ndufa4 and Ndufb6 is the measured expression of change (x-axis) and the significance of the change (y-axis). Groups of genes represented by a single yellow dot share the same change in gene expression compared to their matched controls. Significance is the negative log (base 10) of the p-value, and the most significant changes are plotted higher on the y-axis. The dotted lines represent the thresholds used to select the DE genes: LogFC = 0.6 for expression change and 0.05 for significance (p-value shown in terms of the negative log (base 10) value). B. Gene perturbation bar plot. All the genes in OXPHOS (KEGG: 00190) are ranked based on their absolute perturbation values. The box and whisker plot on the left summarizes the distribution of all the differentially increased genes annotated to this pathway. The box represents the 1st quartile, the median and the 3rd quartile. C. OXPHOS (KEGG: 00190) pathway diagram is overlaid with the computed perturbation of each gene. Perturbation accounts for a gene’s measured fold change and for the accumulated perturbation propagated from any upstream genes (accumulation). The highest positive perturbation is in dark red. Genes are highlighted in all locations in the diagram.</p

Gene expression and mitochondrial function.

A. qRT-PCR confirmed upregulation of the five genes annotated to oxidative phosphorylation in tumors from Hltf-deleted (n = 23) compared to control (n = 14) mice. B. Total cellular ATP was normalized to DNA from Hltf-deleted (n = 8) and control (n = 8) samples that were assayed in duplicate. C. Mouse mitochondrial DNA copy number was determined by the comparison of mitochondrial (mt) and nuclear (n) DNA measured by qPCR. Of the 16 tumor samples from Hltf-deleted mice, the one sample that failed to amplify was excluded from the calculations. Values in each panel are expressed as mean±SEM, ***p≤0.001, **p≤0.01, non-significant (ns) = P>0.05.</p

Histograms of tumor number (A) and gross distribution (B), along with rectal prolapse (C).

Statistical confirmation of the dramatic effect of Hltf-deletion on the total number of tumors (A) and on the spatial distribution of tumors (B) is consistent with the complete protrusion of the rectum through the anal canal beyond the anus (C). Values in panels A and B are mean ± SEM. Values with the same letter designation in panel B are not significantly different.</p

H&E stained Swiss-rolled colons.

The entire large intestine for wild type control (A) and Hltf-deleted (B) mice is shown in each photomicrograph beginning with the cecum in the outmost region of the roll, continuing to the rugated region of the proximal colon, and terminating with the anorectal junction in the innermost region of the roll. Tumors and cellular inflammatory infiltrate in tumor stroma are evident in the distal colon. Pronounced loss of crypt architecture is evident in Hltf-deleted tissue.</p

Macroscopic visualization of tumors in <i>Hltf</i>-deleted colon.

Alcian blue (1%) was topically applied to longitudinally opened colons from two different Hltf-deleted mice to highlight the borders of the individual tumors against the normal epithelium of the colon (upper panel) and each other (lower panel). The heterogeneity of the tumor burden in the distal colon/rectum is well demarcated by the Alcian blue stain.</p

Exploration of the tissue architecture of IC and ID β <i>HLTF</i> KO pancreata.

(A) Two-dimensional batch-corrected t-distributed stochastic neighbor embedding (t-SNE) visualization of the UMI counts from the entire IC vs ID dataset. (B) Pancreata clusters 16 and 18 are shown in t-SNE space. (C) Pancreatic genes insulin 1 (Ins1) and insulin 2 (Ins2) illustrated differential gene expression in IC vs ID β Hltf KO mice in t-SNE plots of clusters 16 and 18. The unique expression of immune cell markers in IC vs ID tissue is elaborated in t-SNE plots of these clusters. Slamf6 and Il2rb were added to previously identified markers for NK cells (GzmA, Klrb1b). Five markers for B cells (Pax5, Blk, Fcmr, Fcrla, Tnfrasf9) and three markers for activation of innate immunity (Bpifb1, Serpinb3a, Defb36) were unique to cluster 18.</p

Differential γH2Ax pan-staining and TUNEL assay.

Abundant γH2Ax in β cells from IC β Hltf KO mice (A) compared to minimal immunostaining in β cells from ID β Hltf KO mice (B). Two-types of γH2Ax pan-staining are evident. β cells from IC β Hltf KO mice have apoptotic rings and the β cells from ID β Hltf KO mice have limited pan-nuclear staining of the entire nucleus. Results from the terminal deoxynuceotidyl transferase dUTP nick-end labeling (TUNEL) assay (C), which detects β cell death-associated DNA fragmentation (3’-OH termini), indicates the amount of DNA damage is more than the targeted β cells can efficiently repair when the animals are IC. Cell-death in IC Hltf +/+ controls and ID β Hltf KO mice was negligible. A positive mouse testis control (D) was included because apoptosis is an important component of normal spermatogenesis.</p