Fig. 5: K. pneumoniae promotes precancerous lesions and HCC in both germ-free and SPF mice.
a, Design of K. pneumoniae gavage for promoting precancerous lesions in germ-free mice without DEN treatment (GF/PBS group n = 10; GF/EC group n = 8; GF/KP group n = 10) and related results (b–h). b, In vivo imaging of body and liver after Cy5.5-d-Lys-labelled K. pneumoniae gavage (left), live bacteria culture of liver tissues on blood agar plate (top right) and Cy3-conjugated EUB338 probe (GF/PBS group), E. coli probe (GF/EC group) or K. pneumoniae probe (GF/KP group) FISH detection in liver tissues (bottom right). c, Gut permeability assays using 500 kDa FITC-dextran (left) (n = 10 (GF/PBS), 8 (GF/EC), 10 (GF/KP)) and quantitative analysis of TEM (middle) (n = 6 biologically independent samples) and E-cad IHC staining (right) (n = 6 biologically independent samples). d, MMP-2/-9 activity in mouse faecal supernatant using gelatin hydrolysis assay (bottom left), gelatin zymography (top left) and dynamic activity assay (right). n = 3 biologically independent samples. e, Masson’s trichrome staining (top left) and COLIV IHC staining (top right) of colon tissues and COLIV protein level by western blot (bottom). n = 6 biologically independent samples. f, Representative images of liver gross morphology (top left) (dashed yellow circles indicate nodules), H&E staining of mice liver sections (bottom left), and nodule number and incidence of dysplasia (right). n = 10 (GF/PBS), 8 (GF/EC), 10 (GF/KP). g, IHC staining for PCNA and Ki-67 (left) (n = 6 biologically independent samples), and western blot assay of PCNA (right) (n = 3 biologically independent samples). h, Desmin (left) and α-SMA (middle) IHC staining, and Sirius red staining (right) of liver sections. n = 6 biologically independent samples. i, Design of K. pneumoniae gavage on precancerous lesions in SPF mice without DEN treatment (PBS group n = 6; EC group n = 6; KP group n = 6) and relevant results (j–n). j, Gut permeability assays using 500 kDa FITC-dextran. n = 6 biologically independent samples. k, Live bacterial culture of liver tissues on blood agar plate (top), and Cy3-conjugated EUB338 probe (PBS group), E. coli probe (EC group) or K. pneumoniae probe (KP group) FISH detection in liver tissues (bottom). n = 6 biologically independent samples. l, TEM (left) and quantitative analysis (right) of colon tissues. n = 6 biologically independent samples. m, Representative images of liver gross morphology (top) (dashed yellow circles indicate nodules), H&E staining of mice liver sections (middle), and nodule number and incidence of dysplasia (bottom). n = 6 biologically independent samples. n, Representative images of IHC staining for PCNA and Sirius red staining of liver sections (left) with quantitative analysis (right). n = 6 biologically independent samples. o, Design of K. pneumoniae gavage on HCC in SPF mice with DEN treatment (DPBS group n = 7; DEC group n = 6; DKP group n = 7) and related results (p). p, Representative images of liver gross morphology (left) (dashed yellow circles indicate tumour) and H&E staining of mice liver sections (middle) with tumour incidence and tumour number quantitative analysis (right). n = 7 (DPBS), 6 (DEC), 7 (DKP). In c (excluding tight junction disappear rate), d, e–h (excluding incidence of dysplasia), j,k,m (excluding incidence of dysplasia), n and p (excluding tumour incidence), data are presented as mean ± s.e.m. Each data point in bar plots represents one mouse. Tight junction disappearance rate, incidence of dysplasia and tumour incidence were analysed using Fisher’s exact test. MMP activity was analysed using two-way ANOVA. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.
