On the following day, HeLa cells were then infected with invasin-expressing E. coli at an MOI of 200 by plate rotation at an rpm of 50, in a 37°C, 5% C02 humidified incubator for 90min.

After infection, HeLa cell were washed twice with phosphoate buffered saline (PBS) and fixed with 3% formaldehyde/ 0.025% glutaraldehyde at room temperature for 10 min.

HeLa cells were then blocked with 5% BSA in PBS for 30 min at room temperature and stained with anti-LPS E. coli (Abcam).

HeLa cells were co-incubated with E. coli

HeLa cells incubated With either control antibody (actin) or antibody against Bl-integrin along With fluorescent secondary antibody (Alexa647).

HeLa cells , infected with E. coli expressing invasin and GFP at an MOI of 200 for 90 minutes, were washed with PBS twice, fixed with paraformaldehyde, and immunostained with an anti-LPS antibody to identify bacterial cell wall (red color) and Hoechst to identify DNA (blue color).

After co-culture of increasing amounts of GFP-expressing E. coli harboring a plasmid permitting arabinose-inducible control of invasin (pBACr-Aralnv) with HeLa cells , flow cytometry was conducted to obtain data shown in Fig 3A.

In the absence of induction, bacteria carrying pBACr-AraIm/ cannot infect host cells, which was reported previously [27] and observed by us by co-incubating HeLa cells with uninduced bacteria (S6A—S6C Fig).

In each experiment, the engineered bacteria were co-cultured with HeLa cells for 90 minutes in well-mixed conditions to mitigate the effects of heterogeneity in the bacterial population.

Consistent with observations reported in the literature [18,22], fluorescence microscopy confirmed drastic cell-cell variability in bacterial uptake by HeLa cells (Fig 1A).

However, immunolabeling experiments revealed a relatively narrow, unimodal distribution of Bl-integrins across HeLa cells (SID Fig).

HeLa cells were treated with indicated compounds at their respective EC90 for 20 hours, fixed with 4% parafor-maldehyde, permeabilized with 0.2% Triton X- 100/ PBS and co-stained for DNA (0.5 ug/ ml Hoechst 33342) and tubulin (rat anti-tubulin primary antibodies and anti-rat Cy3 secondary antibodies).

Consistently, a similar SAR trend was observed by evaluating each compound’s potency (EC50) in HeLa cells with regards to their ability to arrest cells in G2/M-phase and induce cell death.

To test if tubulin was the primary target, we treated HeLa cells with compounds 6—12 and analyzed their effects by IF microscopy.

As expected, compounds 6—12 induced a microtubule depolymerization phenotype in HeLa cells (Figs 5G and 812).

(AF) Immunofluorescence of HeLa cells treated with control DMSO or indicated compounds (1—5) for 20 hours.

(A) For cell Viability assays, HeLa cells were treated with increasing concentrations (20-point titration 0—-100 uM) of indicated compounds (6—12) for 20 hours and the percentage of cells arrested in G2/M was quantified.

Immunofluorescence microscopy of HeLa cells treated with control DMSO, Taxol, col-chicine, or the indicated compounds (6—12) for 20 hours.

The attenuation of the initial response of delayed IEGs to EGF was also shown to depend on negative feedback through de novo transcription in HeLa cells [10], but it is unknown whether negative feedback plays roles in other cell types or under other stimuli.

This set of delayed early genes has been shown to be activated in waves following FOS, JUN and EGRl expression in HeLa cells [10].

This is consistent with reports that NAB2 represses EGR1 and thereby attenuates the immediate-early response in HeLa cells stimulated with EGF [10].