<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

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

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

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

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

Primers for gender identification and genotyping.

Primers for gender identification and genotyping.</p

Photomicrographs of histological sections of mouse placenta on gd18.5.

Sections from wild type (A-E) and Hltf null (F-J) placentae show widespread DBA-lectin reactivity for uNK cells at 10 X magnification (A, F). Comparisons at 40 X magnification of DBA-lectin reactivity for uNK cells (B, G) and amylase-resistant PAS positive uNK cells (C, H) show abundant cytoplasmic granules contained within the cellular membranes. Immunolabeling (20 x magnification) for perforin is negligible in uNK cells from wild type (D; blue arrows) compared with Hltf null (I). Immunolabeling (20 x magnification) for Hltf shows both nuclear and cytoplasmic staining throughout the wild type (E) placenta compared to the complete absence of Hltf protein in the null placenta (J). All insets are negative (minus primary antibody) controls.</p