Intratumoral proliferation of KR-<i>E</i>. <i>coli</i>.

<p><b>a,</b> NPC tumor subcutaneously formed in a nude mouse. <b>b,</b> The distribution of KR-<i>E</i>. <i>coli</i> immediately after intratumoral injection (left), and one day after the injection (right). Scale bar, 5 mm. <b>c,</b> Quantification of the fluorescence intensity of KR-<i>E</i>. <i>coli</i> immediately after the intratumoral injection and one day after the injection (n = 10). *p<0.001 (<i>t</i>-test). Error bars indicate standard error of the mean (SEM). The fluorescence intensity was calculated by summating the digital values of all points in the field of view, and expressed in a relative manner.</p

Growth curves of the HeLa tumors and CNE2 tumors of a large size treated with KR-<i>E</i>. <i>coli</i>.

<p>The black line indicates the volume change of tumors injected with <i>E</i>. <i>coli</i> that expressed no KillerRed. The red dotted line indicates the volume change of CNE2 tumors (volume = 150 mm³) injected with <i>E</i>. <i>coli</i> that expressed KillerRed. The red full line indicates the volume change of HeLa tumors injected with <i>E</i>. <i>coli</i> that expressed KillerRed. All tumors were irradiated at the same dose on the day 7. Before treatment, ^♦p > 0.05 (rank sum test), error bars indicate SEM. After treatment, *P< 0.01 (rank sum test), error bars indicate SEM.</p

Absence of singlet oxygen in the phototoxic effect of illuminated KR-<i>E</i>. <i>coli</i>.

<p>To quantitatively measure the singlet oxygen produced by KillerRed, the intensity of spontaneous luminescence was measured at wavelengths in a near infrared region. (1) KillerRed in PBS. (2) KillerRed in an O₂-saturated solution. (3) KillerRed in a deuterium oxide (D₂O) solution. (4) Methylene blue solution used as a control. In reference to methylene blue, KillerRed did not produce any singlet oxygen upon irradiation.</p

Superoxide is released from KR-<i>E</i>. <i>coli</i> upon orange light irradiation.

<p><b>a,</b> Ordinary light image (left) and fluorescence image (right) of a KR-<i>E</i>. <i>coli</i> suspension observed under a microscope. Bar, 10 m. <b>b,</b> The production of superoxide by the irradiation of KR-<i>E</i>. <i>coli</i> detected with nitroblue tetrazolium (NBT) in an agarose gel. The top panels demonstrate a change in the <i>E</i>. <i>coli</i> with KillerRed before and after irradiation. A magnified portion (right) of the agarose gel near the KR-<i>E</i>. <i>coli</i> containing central pool indicates a formazan precipitation due to the diffusion of superoxide from the pool. Bar, 5 mm. The bottom panels demonstrate change in the color of the <i>E</i>. <i>coli</i> without KillerRed expression before and after irradiation.</p

Histochemistry, and immunohistochemistry staining of xenografts.

<p>A-C: H&E staining of derived xenografts 120 h after inoculation with 1×10⁶ 5-8F cells or primary tumors. Representative images are shown. D-F: IHC staining of human CK34βE12. Magnification, ×400.</p

2D and 3D analysis of invasion and metastasis of NPC cells 48 h after inoculation.

<p>A: Invasion of HONE1-GFP cells through the basement membrane and toward the lower site of CAM, especially in 3D imaging. Invasive depth is shown in the Z-axis. B: Metastasis of HONE1-GFP cells via the blood system. Representative bright-field (DIC), green fluorescent (GFP) and overlay (Merge) images. Magnification, ×40.</p

Quantification of disseminating NPC cells in CAM.

<p>Number of circulating 5-8F and 6-10B cells in the lung and heart of developing chicken evaluated by β-globin–based qPCR and expressed as mean ± SD (n = 5 for each cell line). * <i>P</i><0.05.</p

Tumor xenografts in chorioallantoic membranes (CAMs) inoculated with NPC cells or tumor biopsies.

<p>CAMs inoculated with 0.5×10⁶ or 1×10⁶ 5-8F cells and NPC primary tumors at 24 and 120 h after inoculation. Representative data are shown.</p

Clinical features of NPC patients participated in this study.

<p>^a: NPC patients involved in this study.</p><p>^b: M, male; F, female.</p><p>^c: WHO histopathological classification of NPC (2005), A: keratinizing squamous cell carcinoma; B1: differentiated non-keratinizing carcinoma; B2: undifferentiated non-keratinizing carcinoma; C: Basaloid squamous cell carcinoma.</p><p>^d: The TNM clinical classification for NPC according to AJCC staging, 7th Edition. T-Primary tumor; N-Regional lymph nodes; M-Distant metastasis.</p><p>Clinical features of NPC patients participated in this study.</p

MOESM4 of Engineering Bacillus licheniformis for the production of meso-2,3-butanediol

Additional file 4: Figure S4. Confirmation of the mutant B. licheniformis WX-02Δgdh/pHY-gdh strain by PCR amplification