Additional file 1 of TRAF3 suppression encourages B cell recruitment and prolongs survival of microbiome-intact mice with ovarian cancer
Creators: name: Zorea, Jonathan
name: Motro, Yair
name: Mazor, Roei D.
name: Carmi, Yifat Koren
name: Shulman, Ziv
name: Mahajna, Jamal
name: Moran-Gilad, Jacob
name: Elkabets, Moshe
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Fig. S1. Establishing a library-expressing cells. A WT mouse harboring ITB1-related ascites and small tumors, indicated by the white arrows or the black arrows in the IHC-stained tissues. MLN- mesenchymal lymph node. B Cas9-infected ITB monoclonal isolated colonies express an active (&gt;95%) Cas9. In all flow cytometry experiments, we used a PD-1 guide and determined its expression before and after stimulation with IFN-γ. C Bar graph showing the number of reads per guide. Guides with the same number of reads are grouped together and indicated by the same color. Fig. S2. Knocking out TRAF3 does not affect the NF-κB pathway. A Scratch assay showing the migration of all cells over 48 h into the scratched area. Data was acquired using a live cell imaging system (Julistage). B Western blot of ITB1 and TRAF3KO lysates comparing the levels of proteins of the NF-κB pathway. The levels of p65 and RelB were monitored in the nucleus, and p100 and p52 were monitored in the whole lysates. Actin and histone H3 were used as loading controls. C Quantitative PCR shows the mRNA levels of genes related to the NF-κB pathway (RelB, IL-6, and IκBα). Cyan bars represent ITB1, solid pink bars represent TRAF3KO1, and dotted pink bars represent TRAF3KO2. D MFI of the blot on the main figure, calculated using ImageJ E Bar graph showing the differences in mean fluorescence intensity of the Cy3 channel in the nucleus between ITB1 (cyan) and TRAF3KO cells (pink). Fig. S3. B cells in TRAF3KO-injected mice express and secrete more IgAs than in ITB1-injected mice. A Expression of the costimulatory protein CD40 and interleukin-10 (IL-10) on B cells in ITB1-injected and TRAF3KO-injected mice. B, C Ig isotyping array (B) and its quantification (C). D Scatter plot showing the differentiation between the two clusters of the heatmap. E Heatmap showing the average score of each cluster for each immune pathway. F Survival curve showing the difference between patients with high B cell score (cyan) and low B cell score (dark pink). Fig. S4. Effect of ABX treatment. A Mouse treated with ABX for 10 days and its enlarged cecum. B Fecal samples were collected from both ABX-treated and naïve mice and seeded on a blood agar plate. Growth of colonies can be seen only on the left side of the plate where the naïve cells was seeded. C Difference in the abundance of three genera, compared using the Wilcoxon signed-rank test. Fig. S5. Unique bacteria in the ascites of ovarian cancer patients. A Bar plot showing the log2 fold change (x axis), discovered using Deseq2, of genera expressed at least in one or more patients. Genera expressed more in cirrhosis or OC are in cyan or pink, respectively. Genus with black name are expressed significantly in one of the groups, while the grey ones are not significant. B a schematic illustration describing the separation of IgA-coated bacteria from the ascites fluid. C Bar plot showing the log2 fold change (x axis), discovered using Deseq2, of IgA-coated genera expressed at least in one or more patients. Genera whose expression was higher in cirrhosis or OC are shown in cyan or pink, respectively. Genera written in black are significantly expressed in one of the groups, while those written in gray are not significantly expressed.