clostridium dificile  (ATCC)

Journal: bioRxiv

Article Title: Pancreatic cancer-associated organ dysfunction promotes muscle autophagy and contributes to peripheral tissue wasting

doi: 10.64898/2026.02.27.708635

Figure Lengend Snippet: a. Schematic of experimental design to implant orthotopic murine KP −/− C PDAC cells into the tail of pancreas to form a tumor. b-f. Whole-body weight (b), food intake (c), blood glucose levels (d) and tissue weights normalized to whole-body weights of pancreas (e) and liver (f) 2-weeks after implantation of PBS (control) or pancreatic tumor cells (PDAC) into mice as outlined in a ( n = 4). g. H&E staining of perigonadal AT histology from control and PDAC mice as outlined in a. Adipocyte area was quantified for each image and is also shown ( n = 164 adipocytes counted from n = 4 control mice, and n = 143 adipocytes from n = 4 PDAC mice). h-i. Tissue weights of perigonadal and subcutaneous ATs (h) and quadriceps, gastrocnemius, and soleus muscle (i) normalized to whole-body weights from control and PDAC mice as outlined in a. j. H&E staining of gastrocnemius muscle from control and PDAC mice outlined in a. Myofiber area was also quantified for each image and is also shown. ( n = 386 myofibers counted from n = 5 control and n = 365 myofibers from n = 5 PDAC mice). k. Experimental strategy for administration of uniformly labeled 13 C-glucose (U- 13 C-glucose) or 13 C-starch (U- 13 C-starch) by oral gavage to assess pancreatic exocrine function. l. Fraction of 13 C-labeled glucose (M+6) measured in plasma over time after U- 13 C-glucose administration to 6-week-old control and KP −/− C (PDAC) mice as described in k. ( n = 4). m. Area under curve (AUC) quantified for 13 C-labeled glucose disposal for experiment shown in l. n. Fraction of 13 C-labeled glucose (M+6) measured in plasma over time after U- 13 C-starch administration to 6-week-old control and KP −/− C (PDAC) mice as described in k. ( n = 4). o. Area under curve (AUC) quantified for 13 C-labeled glucose disposal for experiment shown in n. p. Experimental strategy for administration of 15 N- Spirulina diet (contains labeled free amino acids) or 15 N-labeled protein from S. pombe by oral gavage to assess pancreatic exocrine function. q. Fraction of 15 N-labeled alanine (M+1) measured in plasma over time after 15 N- Spirulina diet administration to 6-week-old control and KP −/− C (PDAC) mice as described in p ( n = 4). r. Fraction of 15 N-labeled alanine (M+1) measured in plasma over time after administration of 15 N-labeled protein from S. pombe to 6-week-old control and KP −/− C (PDAC) mice as described in p ( n = 4). s-t. Quantification of total protein amount (s) and protease activity in stool collected from 6-week-old control and KP −/− C mice 6h after 15 N- Spirulina diet gavage as described in p. Protein measured in feces from KP −/− C mice was normalized to that measured in feces from control animals ( n = 8 control, n = 4 KP −/− C PDAC). u. Experimental strategy for administration of olive oil by oral gavage to assess pancreatic exocrine function. v. Plasma triglyceride (TGA) concentration measured 4h after olive oil administration to 6-week-old control and KP −/− C mice as described in u ( n = 5). w. Western blot analysis of the indicted pancreatic enzyme expression in pancreas tissue from 6-week-old control and KP −/− C mice ( n = 4). Total S6 is shown as a loading control. x. Immunohistochemistry (IHC) staining for Pancreatic Lipase (Pl), Trypsin (Prss2), and Alpha-Amylase of pancreas tissue sections from 6-week-old control and KP −/− C mice ( n = 4). Statistical analysis was performed using unpaired two-sided t -tests, data shown are mean ± S.D and n represents the number of mice analyzed. Scale bars: 100 μm for panel g and j; 200 μm for panel x.

Article Snippet: For immunohistochemistry (IHC), the following primary antibodies and dilutions were used: Ki67 (Biocare Medical, CRM325C, clone SP6; 1:50), cleaved Caspase-3 (Cell Signaling Technology, 9664, clone 5A1E; 1:800), PRSS2/trypsin (Abclonal, A19275; 1:100), Pancreatic lipase (PNLIP; Proteintech, 11209-1AP; 1:250; and Abclonal, A6396; 1:250), α-Amylase (Santa Cruz Biotechnology, sc46657, clone G10; 1:250), Insulin (Cell Signaling Technology, 4590; 1:200), Glucagon (Cell Signaling Technology, 2760; 1:200), and CK19 (Abcam, ab133496; 1:200).

Techniques: Control, Staining, Labeling, Starch, Clinical Proteomics, Activity Assay, Concentration Assay, Western Blot, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Pancreatic cancer-associated organ dysfunction promotes muscle autophagy and contributes to peripheral tissue wasting

doi: 10.64898/2026.02.27.708635

Figure Lengend Snippet: a. Schematic showing experimental approach to disrupt autophagy in the muscle of mice with genetically engineered KP −/− F PDAC tumors. b. Western blot assessing Atg7 protein in muscle tissue from 6-week-old control ( Pdx-1-P2A-FlpO; Trp53 Frt/Frt ; Ckm-Cre ) or Atg7 muscle knock-out mice ( Ckm-Cre ; Atg7 fl/fl : referred to as Atg7 mKO), without or with ( Pdx-1-P2A-FlpO; Kras FSF-G12D/+ ; Trp53 Frt/Frt ) KP −/− F PDAC as indicated ( n = 3). Also shown is western blot for Atg7 in pancreas tissue from control and Atg7 mKO mice with KP −/− F PDAC ( n = 5). c. Western blot for Atg7, Atg5, p62, Lc3B-I, and Lc3B-II in gastrocnemius muscle from 6-week-old control and Atg7 mKO mice with KP −/− F PDAC ( n = 2). Vinculin is blotted as a loading control. d. Gastrocnemius, quadriceps, and soleus skeletal muscle weights normalized to whole-body weights from 6-week-old control or Atg7 mKO mice, without or with KP −/− F PDAC as indicated ( n = 14 control, n = 12 Atg7 mKO, n = 16 KP −/− F PDAC, and n = 11 Atg7 mKO KP −/− F PDAC). e. H&E staining of gastrocnemius muscle from 6-week-old control or Atg7 mKO mice, without or with KP −/− F PDAC as indicated. f. Myofiber area quantification from histology shown in e. ( n = 230 control, n = 166 Atg7 mKO, n = 477 KP −/− F PDAC, and n = 186 Atg7 mKO KP −/− F PDAC from n = 4 mice for each group). g-l. Whole-body weight ( n = 14 control, n = 14 Atg7 mKO, n = 16 KP −/− F PDAC, and n = 14 Atg7 mKO KP −/− F PDAC, 6-week-old) (g), food intake ( n = 5) (h), blood glucose ( n = 9) (i), plasma insulin ( n = 7 control, n = 6 Atg7 mKO, n = 5 PDAC, and n = 5 Atg7 mKO KP −/− F PDAC) (j), liver weight ( n = 11 control, n = 12 Atg7 mKO, n = 15 KP −/− F PDAC, and n = 11 Atg7 mKO KP −/− F PDAC) (k), and perigonadal and subcutaneous ATs weights ( n = 14 control, n = 12 Atg7 mKO, n = 16 KP −/− F PDAC, and n = 11 Atg7 mKO KP −/− F PDAC) (l) normalized to whole-body weights of 6-week-old control or Atg7 mKO mice, without or with KP −/− F PDAC as indicated. m. H&E staining of perigonadal AT from control and Atg7 mKO mice with KP −/− F PDAC. n. Pancreas tissue weights normalized to whole-body weights from 6-week-old control or Atg7 mKO mice, without or with KP −/− F PDAC as indicated ( n = 11 control, n = 12 Atg7 mKO, n = 11 KP −/− F PDAC, and n = 12 Atg7 mKO KP −/− F PDAC). o. Gross images of pancreas from 6-week-old control and Atg7 mKO mice with KP −/− F PDAC as indicated ( n = 2). p. H&E staining of pancreas tissue from 6-week-old control or Atg7 mKO mice, without or with KP −/− F PDAC as indicated. q. Survival of control mice with KP −/− F PDAC ( Pdx-1-P2A-FlpO; Kras FSF-G12D/+ ; Trp53 Frt/Frt , referred to here as KP −/− F PDAC), or the same mice with a Ckm-Cre and one ( Atg7 fl/+ ) or two ( Atg7 fl/fl ) Atg7 fl alleles as indicated ( n = 43 PDAC, n = 50 Atg7 fl/+ , and n = 82 Atg7 fl/fl ). r. Survival of control or Atg7 mKO mice following orthotopic implantation of murine KPC PDAC cells into pancreas ( n = 6). s. Survival analysis of control mice and mice with muscle-specific loss of function of Atrogin-1 ( Atrogin-1 fl/fl ; Ckm-Cre: Atrogin-1 mLOF) following orthotopic pancreatic implantation of KPC PDAC cells ( n = 6). t. Survival analysis of control mice and mice with combined muscle-specific Atrogin-1 loss of function and Atg7 deletion ( Atrogin-1 mLOF; Atg7 mKO) following orthotopic KPC PDAC implantation ( n = 6). Both male and female mice were used in experiments. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test for data in panels c-j and m-n. P-values on survival experiments were calculated with Gehan-Breslow-Wilcoxon test. Data are presented as mean ± S.D., and n denotes the number of mice analyzed. Scale bars: 100 μm.

Article Snippet: For immunohistochemistry (IHC), the following primary antibodies and dilutions were used: Ki67 (Biocare Medical, CRM325C, clone SP6; 1:50), cleaved Caspase-3 (Cell Signaling Technology, 9664, clone 5A1E; 1:800), PRSS2/trypsin (Abclonal, A19275; 1:100), Pancreatic lipase (PNLIP; Proteintech, 11209-1AP; 1:250; and Abclonal, A6396; 1:250), α-Amylase (Santa Cruz Biotechnology, sc46657, clone G10; 1:250), Insulin (Cell Signaling Technology, 4590; 1:200), Glucagon (Cell Signaling Technology, 2760; 1:200), and CK19 (Abcam, ab133496; 1:200).

Techniques: Western Blot, Control, Knock-Out, Staining, Clinical Proteomics

Journal: bioRxiv

Article Title: Pancreatic cancer-associated organ dysfunction promotes muscle autophagy and contributes to peripheral tissue wasting

doi: 10.64898/2026.02.27.708635

Figure Lengend Snippet: a-c. Volcano plot of log2 fold change (logFC) in gene expression in PDAC tumors isolated from 6-week-old mice with KP −/− F PDAC ( Pdx-1-P2A-FlpO; Kras FSF-G12D/+ ; Trp53 Frt/Frt ) without or with Atg7 ( Pdx-1-P2A-FlpO; Kras FSF-G12D/+ ; Trp53 Frt/Frt ; Ckm-Cre; Atg7 fl/fl , ( Atg7 mKO KP −/− F PDAC)) loss in muscle for genes involved in amino acid (a), glucose (b), and cholesterol metabolism (c). ( n = 5). d. Schematic showing experimental design for use of diets with different amounts of protein or different amounts of free amino acids instead of protein test effects on mice with KP −/− F PDAC and Atg7 mKO KP −/− F PDAC. e-g. Whole-body weights (e), liver weights (f), and pancreatic tumor weights (g) normalized to total body weight measured at endpoint after 4-week-old mice were fed diets with different free amino acid (FAA) amounts as per the experiment outlined in d ( n = 8 Atg7 mKO KP −/− F PDAC on 0.5X protein, n = 7 Atg7 mKO KP −/− F PDAC on 1X FAA, and n = 8 Atg7 mKO KP −/− F PDAC on 2X FAA). h. Gross image of dissected pancreas following 2-weeks of the indicated FAA diet administration as outlined in d. i-j. Perigonadal and subcutaneous ATs (i), quadriceps, gastrocnemius, and soleus muscle (j) weights normalized to total body weight measured at endpoint after 4-week-old mice were fed diets with different FAA amounts as per the experiment outlined in d ( n = 8 Atg7 mKO KP −/− F PDAC on 0.5X FAA, n = 7 Atg7 mKO KP −/− F PDAC on 1X FAA, and n = 8 Atg7 mKO KP −/− F PDAC on 2X FAA). k. Ki-67 and cleaved Caspase-3 IHC to assess proliferation and apoptosis, respectively, in Atg7 mKO KP −/− F PDAC tumor tissue after 2-weeks of the indicated FAA diet administration as outlined in d ( n = 3). l. Survival of Atg7 mKO KP −/− F PDAC mice fed diets with different amounts of intact protein as described in d ( n = 16 Atg7 mKO KP −/− F PDAC on 0.5X protein, n = 9 Atg7 mKO KP −/− F PDAC on 1X protein, and n = 8 Atg7 mKO KP −/− F PDAC on 2X protein diet). m . Survival of Atg7 mKO KP −/− F PDAC mice fed diets with different amounts of FAAs as described in d ( n = 16 Atg7 mKO KP −/− F PDAC on 0.5X FAA, n = 14 Atg7 mKO KP −/− F PDAC on 1X FAA, and n = 12 Atg7 mKO KP −/− F PDAC on 2X FAA). Both male and female mice were used in these experiments. P-values on survival experiments in l-m were calculated with Gehan-Breslow-Wilcoxon test. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test for data in other panels, data are mean ± S.D and n represents the number of mice analyzed. Scale bars: 200 μm.

Article Snippet: For immunohistochemistry (IHC), the following primary antibodies and dilutions were used: Ki67 (Biocare Medical, CRM325C, clone SP6; 1:50), cleaved Caspase-3 (Cell Signaling Technology, 9664, clone 5A1E; 1:800), PRSS2/trypsin (Abclonal, A19275; 1:100), Pancreatic lipase (PNLIP; Proteintech, 11209-1AP; 1:250; and Abclonal, A6396; 1:250), α-Amylase (Santa Cruz Biotechnology, sc46657, clone G10; 1:250), Insulin (Cell Signaling Technology, 4590; 1:200), Glucagon (Cell Signaling Technology, 2760; 1:200), and CK19 (Abcam, ab133496; 1:200).

Techniques: Gene Expression, Isolation

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A) QTRT1 expression was significantly downregulated in colonic epithelial cells from patients with Crohn’s Disease, compared with healthy controls as determined by scRNA-seq. The data were retrieved from NCBI GEO scRNA-seq database (accession GSE164985), which included 43,692 cells from three CD patients and 51,036 cells from four healthy controls. Data are shown as mean ± SD, Welch’s t-test. (B) Q-associated bacteria Bacteroides (k=9 datasets) and (C) Alistipe (k=8 datasets) were significantly reduced in IBD patients compared with healthy controls at species level, based on analyses of publicly available human gut metagenomic datasets. Data are expressed as means ±SD, Wilcoxon rank sum test. (D) QTRT1 -/- mice exhibited 12 unique OTUs of microbiome, compared to the wild-type mice, based on 16s rRNA sequencing of the fecal samples. There are significant differences in bacteria: decreased Bacteroides and Alistipe, and increased Akkermansia , compared with Wildtype mice. ( E) Decreased Bacteroides and ( F ) increased Akkermansia in QTRT1 -/- mice was determined by RT-PCR. Data are expressed as the mean□±□SD. Wildtype mice n=10, QTRT1 -/- mice n=5. Welch’s t- test. (G) Akkermansia in the colons of QTRT1 -/- and Wildtype mice was detected by fluorescence in situ FISH using the Akkermansia -specific Cy3-labeled probe ( AKK -Cy3). (H) Quantification of the Cy3-positive area (in pixels) from FISH images. n□=□3 mice/genotype. Welch’s t- test. (I) Bacteria in the colon of QTRT1 -/- and Wildtype mice were found by fluorescence in situ hybridization. (J) Quantification of the EUB338-positive area (in pixels) from FISH images. n□=□3 mice/genotype. The red dashed line outlines the epithelial surface. The scale bar is 20□μm. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Expressing, Bacteria, Sequencing, Reverse Transcription Polymerase Chain Reaction, Fluorescence, In Situ, Labeling, In Situ Hybridization

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A) Mucus thickness in the colon tissues of QTRT1 -/- and Wildtype mice was evaluated by FISH staining with EUB338 for all the bacterial species; the scale bar is 5□μm. (B) Quantification of mucus layer thickness in the colon. Three points were randomly selected for each mouse. n□=□10. (C) Representative images of Alcian Blue/PAS staining of the colon of QTRT1 -/- and Wildtype mice. The scale bar is 200□μm. Images are from a single experiment and represent three mice per group. Four crypts were randomly selected for each mouse. Data are expressed as mean ±SD., n = 12 for Wildtype, and n = 10 for QTRT1 -/- , Welch’s t- test. (D) Representative confocal images of colonic tissues from wildtype and QTRT1 -/- mice stained for Mucin2 and DAPI. Data are expressed as mean ±SD, n = 3, Welch’s t- test. (E) Representative immunofluorescence staining of small intestinal sections from Wildtype and QTRT1 -/- mice. Sections were stained for Paneth cell marker Lysozyme (red). (F) Quantification of Lysozyme-positive cells per crypt shows a significant reduction in Paneth cell numbers in QTRT1 -/- mice. Four crypts were randomly selected for each mouse. Data are expressed as mean ±SD., n = 12, Welch’s t- test. (G) Number of lysozyme-positive granules per crypt demonstrating a significant decrease in Paneth cell granule content in QTRT1 -/- mice. Images are from a single experiment and represent three mice per group. Four crypts were randomly selected for each mouse. Data are expressed as mean ±SD., n = 12, Welch’s t- test. (H and I) Western Blot of colonic epithelial cell lysates from Wildtype and QTRT1 -/- mice, showing the expression of CDC42, CD14, CD4, and TNF-α. Villin was used as a loading control. Data are expressed as mean ± SD. n = 3 mice/genotype, Welch’s t- test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Staining, Immunofluorescence, Marker, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A, B) Western blot analysis of tight junction proteins in colonic tissues from Wildtype and QTRT1 -/- mice. QTRT1 deficiency markedly decreased ZO-1 expression, while Claudin-2 and Claudin-10 levels were significantly increased. Claudin-7 remained unchanged. Villin was used as a loading control. Data are expressed as mean ± SD. n = 3 mice/genotype, Welch’s t- test. Immunofluorescence staining of (C and D) ZO-1 in colon tissues showed disrupted and discontinuous ZO-1 localization in QTRT1 -/- mice. (E and F) Claudin-7 (yellow) distribution remained intact in both wildtype and QTRT1 -/- mice, and (G and H) Claudin-10 (magenta) showed a prominent increase at the apical membrane of colonic epithelial cells in QTRT1 -/- mice. Nuclei are stained with DAPI (blue). The scale bar is 20□μm. All data are expressed as mean ± SD, Welch’s t- test, n = 3 mice/genotype. (I) QTRT1 -/- mice had significantly higher intestinal permeability levels compared to Wildtype mice. All data are expressed as mean ± SD, Welch’s t- test, n = 6 mice/genotype. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Membrane, Permeability

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A) Schematic illustration of the generation of QTRT1 ΔIEC mice. QTRT1 LoxP mice (C57BL/6 background) were crossed with Villin-Cre mice to achieve intestine epithelial-specific deletion of QTRT1 in exons 1-3. (B and C) Western blot showed QTRT1 protein levels in isolated intestinal epithelial cells from QTRT1 LoxP and QTRT1 ΔIEC mice. Villin was used as a loading control. Data are expressed as mean ± SD. n = 3 mice/genotype, Welch’s t- test. (D) Quantification of the EUB338-positive area (in pixels) from FISH images. n□=□3 mice/genotype. The dashed red line denotes the mucus layer boundary. (E) Quantification of mucus thickness in colon sections. Data are expressed as mean ± SD. n = 10, Welch’s t- test. (F) Quantification of EUB338-positive bacterial area in colon sections. Data are expressed as mean ± SD. n = 5, Welch’s t- test. (G and H) Immunofluorescence staining of ZO-1 in colon tissues showed disrupted and discontinuous in QTRT1 ΔIEC mice. The scale bar is 20 µm. Data are expressed as mean ± SD. n = 5, Welch’s t- test. (I and J) Western Blot of ZO-1 in IECs from QTRT1 ΔIEC mice and QTRT1 LoxP mice, with Villin as a loading control. Data are expressed as mean ± SD. n = 3, Welch’s t- test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Western Blot, Isolation, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A and B) Immunofluorescence staining of colonic sections from Wildtype and QTRT1 -/- mice showing colocalization of QTRT1 -/- with the mitochondrial marker Tomm20 (red), QTRT1, and nuclei stained with DAPI (blue). The scale bar is 50□μm. Data are expressed as mean ± SD. n = 3 mice/genotype, Welch’s t- test. (C and D) ATP staining shows decreased mitochondrial ATP production in colonic sections of QTRT1 -/- mice compared to Wildtype controls. The scale bar is 50□μm. Data are expressed as mean ± SD. n = 3 mice/genotype, Welch’s t- test. (E) Quantification of reactive oxygen species (ROS) in colon and ileum tissue lysates from Wildtype and QTRT1 -/- mice. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. (F and G) Immunofluorescence staining for Cytochrome C and Tomm20 (red) in colonic tissue. DAPI stains nuclei. The scale bar is 20□μm. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. (H and I) Western blot analysis of Cytochrome C levels in cytosolic and mitochondrial fractions from colonic epithelial cells. COX IV serves as the mitochondrial loading control. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Control

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A) QTRT1 -/- mice display disrupted and fragmented mitochondrial networks along with increased cytoplasmic and extracellular mtDNA release by Immunofluorescence staining of colonic sections from Wildtype and QTRT1 -/- mice using Tomm20 (red) and mtDNA antibodies. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. (B) Increased Cleaved Caspase-3 expression and DNA in the colonic epithelium of QTRT1 -/- mice by immunofluorescence staining. The scale bar is 20□μm. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. (C, D) Western blot analysis of colon tissue lysates of increased Cleaved caspase-3, Bax, phosphorylated PARP (p-PARP), decreased expression of BCL-2, and increased BAX/BCL-2 in QTRT1 -/- mice. And the increase in Cleaved caspase-1 (P20/P22) levels. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. (E and F) A marked increase in TUNEL-positive cells in QTRT1 -/- mice compared to wild-type controls by TUNEL staining of colon sections. The scale bar is 20□μm. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. (G and H) The numbers of proliferating epithelial cells were observed by immunofluorescence staining of PCNA in colonic crypts. Nuclei are stained with DAPI (blue). The scale bar is 20□μm. All data are expressed as mean ± SD, n = 3 mice/genotype, Welch’s t- test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, TUNEL Assay

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A) Western blot confirming reduced QTRT1 protein levels in QTRT1-KD CaCO-2 cells compared to control. All data are expressed as mean ± SD, n = 3 independent experiments, Welch’s t -test. (B and C) Mitotracker-red staining shows fragmented and disorganized mitochondrial networks in QTRT1 KD cells. The scale bar is 5□μm. All data are expressed as mean ± SD, n = 3 individual experiments, each performed in triplicate. Welch’s t -test. (D and E) ATP generation from mitochondria stained by Tomm20 (red) was tested by immunofluorescence staining in QTRT1-KD CaCO-2 cells and Scramble CaCO-2 cells controls. The scale bar is 10 μm. All data are expressed as mean ± SD, n = 4 individual experiments, each performed in triplicate. Welch’s t -test. (F) MitoSox-Red staining reveals elevated mitochondrial ROS levels in QTRT1-KD cells by Flow cytometry. All data are expressed as mean ± SD, n = 3 individual experiments. Welch’s t -test. (G, H, and I) Flow cytometry was used to investigate mitochondrial membrane potential (MMP) and depolarized cells with JC-10 assays. All data are expressed as mean ± SD, n = 3 individual experiments. Welch’s t -test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Western Blot, Control, Staining, Immunofluorescence, Flow Cytometry, Membrane

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A and B) Decreased expression of mitochondrial markers Tomm20 and mitochondrial Cytochrome C, accompanied by increased cytoplasmic Cytochrome C, in QTRT1 KD CaCO-2 cells was tested by Western Blot. And upregulation of Cleaved Caspase-3 and Cleaved Caspase-1 was detected by Western blot analysis. All data are expressed as mean ± SD, n = 3 individual experiments. Welch’s t -test. (C and D) Flow cytometry analysis reveals a higher proportion of apoptotic cells in QTRT1 KD compared to control cells. All data are expressed as mean ± SD, n = 3 individual experiments. Welch’s t -test. (E-G) Mtphagy dye and lysosome dye co-staining show increased mitophagy activity in QTRT1-KD cells. The scale bar is 5□μm. All data are expressed as mean ± SD, n = 3 individual experiments. Welch’s t -test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Expressing, Western Blot, Flow Cytometry, Control, Staining, Activity Assay

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A) Schematic overview of human colon organoid generation. (B) Representative immunofluorescence staining of colon organoids with Tomm20 and QTRT1. ( C ) QTRT1 (green) and (D) Mitochondrial marker Tomm20 (red) fluorescence area per crypt, significant reduced in epithelial in IBD patient organoids. The scale bar is 50□μm. All data are expressed as mean ± SD, n = 3 patients. Three crypts were randomly selected for each organoid. Welch’s t -test. (E) Representative brightfield and Mitotracker-red imaging of colon organoids derived from health control and IBD patients. All data are expressed as mean ± SD, n = 3 patients, Welch’s t -test. (F and G) Quantification of Mitotracker-red integrated density and red fluorescence area per crypt. All data are expressed as mean ± SD, n = 3 patients. In Figure G , three crypts were randomly selected for each organoid. Welch’s t -test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Imaging, Derivative Assay, Control

Journal: bioRxiv

Article Title: Disrupted tRNA modification leads to intestinal mitochondrial dysfunction and microbial dysbiosis

doi: 10.1101/2025.11.27.690007

Figure Lengend Snippet: (A). 10 μM Mito-Q treatment of normal and IBD colon organoids for 24 h. Representative immunofluorescence images of colon organoids labeled for QTRT1 and Tomm20 (red), with merged images including nuclei (blue). (B) Quantification of Tomm20 red fluorescence area and (C) QTRT1 green fluorescence per crypt. The scale bar is 50□μm. All data are expressed as mean ± SD, the data points present 3-4 organoids for each patient. Quantification was performed using three crypts per patient, with three patients per group, n = 3 patients. Welch’s ANOVA test was used in (B) and (C), followed by a post-hoc test using the Dunnett’s T3 multiple comparation test. All P -values are shown in the figures.

Article Snippet: QTRT1 LoxP mice were generated via the CRISPR/Cas9/Cre method by Cyagen Biosciences (Santa Clara, CA, USA) in the C57BL/6 mouse strain background.

Techniques: Immunofluorescence, Labeling, Fluorescence