Additional file 1 of Interplay of gut microbiota and host epithelial mitochondrial dysfunction is necessary for the development of spontaneous intestinal inflammation in mice
Creators: name: Alula, Kibrom M.
name: Dowdell, Alexander S.
name: LeBere, Brittany
name: Lee, J. Scott
name: Levens, Cassandra L.
name: Kuhn, Kristine A.
name: Kaipparettu, Benny A.
name: Thompson, Winston E.
name: Blumberg, Richard S.
name: Colgan, Sean P.
name: Theiss, Arianne L.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Neuroscience
subject: Chemical Sciences not elsewhere classified
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Additional file 1: Fig. S1. Elimination of gut microbiota by broad-spectrum antibiotics (ABX) prevents the manifestation of crypt base AB-PAS+cells in Phb1 deficient mice. (A) Bacterial elimination by ABX shown by reads per sample by 16S rRNA sequencing. (B) AB-PAS staining of ileum. Bar = 100 μm. Dashed line denotes crypt base. (C) Number of AB-PAS+cells/crypt base across 50 crypts.AB-PAS, alcian blue-periodic acid Schiff. n = 7-13 each group. ****P&lt; 0.0001. Fig. S2. Co-housed Phb1ΔPC mice exhibit similar ileal microbiota composition as Phb1fl/fl littermates.16S RNA sequencing of luminal ileal content of co-housed Phb1fl/fl and Phb1ΔPC mice. (A) Alpha diversity measure by Shannon index. (B) Weighted UniFrac PCoA plot of 16S rRNA gene sequences. (C-D) Relative abundance of bacteria at phylum level (C) in individual mice and (D) combined mice by genotype. (E-F) Relative abundance of bacteria at genus level (E) in individual mice and (F) combined mice by genotype. n = 9 each genotype. Fig. S3. Co-housed Phb1ΔPCmice manifest PC abnormalities and ileitis. (A) H&amp;E staining showing Paneth cells (pink granules) in the ileal crypts. Bar = 50 μm; boxed pullout bar = 200 μm. (B) Immunofluorescent-staining for lysozyme (red), muc2 (green), and DAPI (nucleus, blue) in ileal crypts (dashed line). Arrows denote Paneth cells with normal lysozyme packaging into granules. Bar = 50 μm. (C) Average number of lysozyme+cells per crypt per mouse. A minimum of 50 crypts per mouse were quantitated. n = 13 Phb1ΔPCor 10 Phb1fl/fllittermates. (D) Paneth cell lysozyme allocation patterns. A minimum of 50 crypts per mouse were quantitated. n = 13 Phb1ΔPCor 10 Phb1fl/fllittermates. (E) mRNA quantification in ileum by qRT-PCR. n= 14 each genotype. (F) AB-PAS staining of ileum and number of AB-PAS+cells/crypt base. Bar = 100 μm. Dashed line denotes crypt base. n = 13 Phb1ΔPCor 10 Phb1fl/fllittermates. (G) Representative H&amp;E-stained ileum and histological inflammation scoring of ileum. Bar = 100 μm. n= 21 Phb1ΔPCor 10 Phb1fl/fllittermates. (H) mRNA quantification in ileum by qRT-PCR. n = 14 each genotype. AB-PAS, alcian blue-periodic acid Schiff; Ang4, angiogenin 4; H&amp;E, hematoxylin &amp; eosin; Ifng, interferon gamma; Il1b, interleukin 1 beta; muc2, mucin2; Tnfa, tumor necrosis factor alpha. Results are presented as individual mice ±SD. *P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.001, ****P&lt; 0.0001. ****P&lt; 0.0001. Fig. S4. Phb1iΔIECmice co-housed with Phb1fl/fllittermates exhibit Paneth cell defects and spontaneous ileitis. (A) H&amp;E staining showing Paneth cells (pink granules) in the ileal crypts. Bar = 50 μm. (B) Immunofluorescent-staining for lysozyme (red), muc2 (green), and DAPI (nucleus, blue) in ileal crypts (dashed line). Arrows denote Paneth cells with normal lysozyme packaging into granules. Bar = 50 μm. (C) Average number of lysozyme+cells per crypt per mouse. n = 14 Phb1fl/fland 18 Phb1ΔPCmice. (D) Paneth cell lysozyme allocation patterns. n = 16 each genotype. (E)mRNA quantification in ileum by qRT-PCR. n =15 Phb1fl/fland 14 Phb1ΔPCmice.Outliers were identified by ROUT test (Q = 1%) and were removed fromPhb1fl/flAng4and Phb1ΔPCCryptdin3, Cryptdin 5. (F) AB-PAS staining of ileum and number of AB-PAS+cells/crypt base. Bar = 100 μm. Dashed line denotes crypt base. n= 15 each genotype. (G) Histological inflammation scoring of ileum. Arrow denotes infiltrating immune cells. Bar = 100 μm. n = 15 each genotype. AB-PAS, calcian blue-periodic acid Schiff; Ang4, angiogenin 4; H&amp;E, hematoxylin &amp; eosin; muc2, mucin2. Results are presented as individual mice ±SD. **P&lt; 0.01, ***P&lt; 0.001, ****P&lt; 0.0001. Fig. S5. Butyrate supplementation prevents Paneth cell defects in Phb1-deficient mice. (A-B) Immunofluorescent-staining for lysozyme (red), muc2 (green), and DAPI (nucleus, blue) in ileal crypts (white outline) of Phb1ΔPCmice and Phb1fl/fllittermates (A) or Phb1iΔIECmice and Phb1fl/fllittermates (B). Arrows denote Paneth cells with normal Lysozyme packaging into granules. Asterisk denotes Muc2 colocalization with Lysozyme. Bar = 50 μm. (C-D)AB-PAS staining of ileum. Bar = 100 μm. Dashed line denotes crypt base. AB-PAS, alcian blue-periodic acid Schiff; muc2, mucin2; Veh, vehicle. Fig. S6. Butyrate protects Phb1-deficient ileal enteroids from Phb1iΔIEC microbiota-induced death. (A) Western blots for total and cleaved Caspase 3 indicating apoptosis. (B) Oxygen consumption relative to Phb1fl/flvehicle enteroids. As a positive control, enteroids were treated with 0.1 μM Antimycin A 15 min prior to oxygen measurements. n = 3 for all treatments. (C) Mitochondrial superoxide level as measured by MitoSOX fluorescence intensity. n = 3 for all treatments, results are representative of 2 separate experiments. C: Phb1fl/fl microbiota filtrate, KO: Phb1iΔIEC microbiota filtrate. n = 3 for all treatments. AntiA, Antimycin A. Results are presented as individual mice ± SD. *P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.005, ****P&lt; 0.0001.