Figure Legends
Figure 1- (A) The overall structure of the construct co-expressing the cardiac peptides and GFP under the control of synthetic nirB promoter. (B) results of GFP and Cardiac peptide PCR. Lane 1: the DNA size marker, Lane 2: result of GFP PCR, Lane 3: result of cardiac peptides PCR, Lane 4: Negative control (C ) Result of SOEing PCR. Presence of a single band at 550 bp in electrophoresis confirmed correct performance of SOEing PCR. Lane 1: the DNA size marker, Lane 2: result of SOEing PCR Lane 3: Negative control(D) Results of colony PCR, confirming the presence of constructs in recombinant bacteria. Lane 1: The negative control (prepared PCR mixture applying water instead of DNA template), Lane 2: the DNA size marker, Lane 3: the positive colonies comprising the cardiac hormone expressing construct.
Figure 2- Results of SDS-PAGE analysis. (A) Presence of a single band at about 5 kDa confirmed expression of cardiac peptides. Lane 1: protein marker, Lane 2: before induction of hypoxic condition, Lane 3: 24 h after induction of hypoxic condition.(B) The single band at 27 kDa relates to the molecular weight of GFP which is absent in the bacterial samples non-transformed with the cassette. Lane 1: before induction of hypoxic condition, Lane 2: protein marker, Lane 3: 24 h after induction of hypoxic condition.
Figure 3- (A) In vitro  cytotoxicity of purified cardiac peptides from 48h growing media of bacteria bearing construct under hypoxic condition. (B) The results of Annexin-V/7-AAD flow cytometry analysis. Treating MCF-7 cells with cardiac peptides mixture significantly increased rate of apoptosis compare to PBS receiving group (Left: PBS, Middle: media of bacteria without construct, Right: cardiac peptides purified from media of bacteria bearing construct).
Figure 4- (A) Bacterial concentration in tumor tissue and liver of mice 48 h after IV administration of 108 cfu/g bacteria. Accumulation in tumor site was about 15-fold greater compare to the liver. (B) Results of In vivo fluorescence reflectance imaging 0, 1, 3 and 6 days post injection. on day 6 post injection, Maximum New fluorescent emissions were completely restricted to the tumor site and reached to its maximum on day 6 post injection.
Figure 5- (A) suppression of tumor growth by administration of different concentrations of bacteria bearing construct. Increase in concentration of administered bacteria significantly enhanced tumor suppression potency. Ns: not significant, ****: P<0.0001 compared to PBS, ####: P<0.0001 compared to bacteria bearing construct at concentrations equal to 107 cfu/g(B) Results of Kaplan-Meyer analysis demonstrating a higher rate of survival in mice receiving bacteria bearing construct compared to PBS and bacteria without construct.
Figure 6- Alteration in expression of pro-inflammatory cytokines in response to bacterial therapy. Results are expressed as fold changes compared to control group receiving PBS. *: P<0.05, **: P<0.01, ***: P<0.001 compared to control group.
Figure 7- Results of immunohistochemical analysis, quantification of micro-vessel density and H&E staining.