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: Membranes were blocked in 5% non-fat milk in TBS-T and probed overnight at 4 °C with the appropriate antibody diluted in 5% BSA in TBS-T. Primary antibodies were used including Vinculin (Cell Signaling Technology, 13901, clone E1E9V, 1:1,000), PRSS2 (Trypsin, Abclonal, A19275, 1:1,000), PNLIP (Pancreatic lipase, 11209-1AP-Proteintech, 1:1,000), Elastase (Abcam, ab21590, 1:1,000 dilution), α-Amylase (Santa Cruz, G10 clone-sc46657, 1:1,000), LC3B (Cell Signaling Technology, E7X4S, 1:1,000), phospho-ribosomal protein S6 Ser 235/236 (Cell Signaling Technology, 4858, 1/1,000), ribosomal protein S6 (Cell Signaling Technology, 2217, 1:1,000), phospho-p70 S6 kinase Thr389 (Cell Signaling Technology, 9205, 1:1,000), p70 S6 kinase (Cell Signaling Technology, 9202, 1:1,000), phospho-AMPK (Cell Signaling Technology, 2535, 1:1,000), AMPK (Cell Signaling Technology, 2532, 1:1,000), FOXO1 (Abclonal, A13862, 1:1,000), FOXO3A (Abclonal, A0102, 1:1,000), Anti-HA (Millipore-Sigma, 11867423001, 1:1,000), Cathepsin B (Cell Signaling Technology, D1C7Y, 1:1,000), Cathepsin L (R&D Systems, AF1515-SP, 1:1,000), ULK1 (Cell Signaling Technology, D8H5, 1:1,000), ATG5 (Cell Signaling Technology, D5F5U, 1:1,000), ATG7 (Sigma-Aldrich, A2856, 1:1,000), Puromycin (Sigma, MABE343, 1:10,000), EIF3F (Fortis Life Sciences, A303-005A-T, 1:1,000), Proteasome 20S Alpha7 subunit (ENZO Life sciences, BML-PW8110-0025, 1:1,000), Proteasome 19S Rpt3/S6b subunit (ENZO Life sciences, BML-PW8765-0025, 1:1,000), UBCJ2 (ENZO Life sciences, ENZ-ABS840-0100, 1:5,000), BNIP3 (Abcam, ab109362, 1:1,000), phosho-Acetyl-CoA Carboxylase I (S79) (Cell Signaling Technology, 6571S, 1:1,000), Acetyl-CoA Carboxylase I (Cell Signaling Technology, 3661S, 1:1,000), ATG3 (Cell Signaling Technology, 3415, 1:1,000), Calpain (Thermo Scientific, 9A4H8D3, 1:1,000), Lamp1 (Cell Signaling Technology, C54H11, 1:1,000), Lamp2 (Santa Cruz, H4B4, sc-18822, 1:1,000), Catalase (Cell Signaling Technology, D4P7B, 12980S, 1:1,000), Histone H3 (Cell Signaling Technology, D1H2, 4499T, 1:1,000), Cox IV (Cell Signaling Technology, 3E11, 4850T, 1:1,000), MuRF1 (ECM Biosciences, MP3401, 1:1,000), Atrogin-1 (ECM Biosciences, A2041P, 1:1,000).

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