Extended Data Fig. 1: Increasing intracellular STm load over time co-occurs with dynamic proteome changes.
a) RAW264.7 cells infected with STm constitutively expressing mCherry (pFCcGi) at MOI 100:1 and incubated for the indicated times were analysed by CellProfiler. SPI-1 OFF bacteria were chosen to mimic the pathogen-host interaction at systemic sites. After bacterial internalization, cells were treated with gentamicin to kill non-internalised bacteria, synchronise the infection and avoid re-infection cycles. The intracellular bacterial load was quantified at the single-cell level to ensure sampling times spanned distinct phases of intracellular STm proliferation states i.e. pre-proliferation (4h), initial (8h) and extensive (20h) proliferation post uptake. Images were captured at 20x objective and analysis was conducted with CellProfiler to segment infected from uninfected cells and quantify bacterial load per cell based on mCherry fluorescence per infected cell. CellProfiler quantification of bacterial count per infected cell (only infected cells) are displayed as a histogram and shows increasing bacterial load with time. Data contains combined counts from two biological replicate experiments, whereby each replicate received reverse SILAC labels (see Methods). 2-5 fields of view (technical replicates) were acquired per biological replicate and per time point. The combined total of infected host cells quantified is indicated (n=). The dotted red line indicates distribution median. Representative images of quantified cells are displayed to the right of histograms. Scale bars represent 50 μm. The % of infected cells per field of view (FOV) at the beginning of the experiment (i.e. immediately post gentamicin 100 μg/ml treatment, t=0) is displayed as a boxplot on the far right. Box plots are depicted as in Fig. 3C. n denotes FOV, amounting to 1224 cells in total (infected & non-infected). b) Replicate correlation of infected versus uninfected samples for the indicated time points and different subcellular fractions - cytoplasmic and solubilised organelles within the Tx-100 soluble fraction (lysatome; upper), nuclear enriched fraction in the Tx-100 insoluble fraction (nucleome-middle) and proteins secreted into the culture supernatant (secretome-lower). Two biological replicates containing reversed SILAC labels were obtained for each cell fraction and each time point (See Supplementary Table 1). We observed concordant biological replicate correlation (r = Pearson’s correlation) across all time points, and within each subcellular fraction (mean r = 0.886). Data is from n=2 biologically independent samples. A red dotted line represents a linear model by robust regression and r refers to the Pearson’s correlation coefficient.
