Additional file 4: Figure S2A. of Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems

According to gastric cancer disease stage, ERBB2 expression in Asian (GEO accession: GSE36968) and Caucasian (GEO accession: GSE63288) datasets. a Box plots depicting ERBB2 expression in the Asian dataset (GEO accession: GSE36968). The x-axis represents stages and the y-axis is the logarithm of RPKM (a measure of gene expression). b Box plots depicting ERBB2 expression in the Caucasian dataset (GEO accession: GSE63288). The dataset did not contain stage IV patients. The x-axis represents GC disease stages and the y-axis is the logarithm of RPKMs. (DOC 88 kb

Additional file 1: Figure S1A. of Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems

Of 38 gastric cancer cell lines, we found that NCI-N87 cells, the basis for most preclinical anti-ERBB2 studies, had the highest ERBB2 expression level. (DOC 152 kb

Additional file 3: Table S2. of Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems

Stage comparison according to ERBB2 expression in Asian (GEO accession: GSE36968). For comparisons, we used one-way ANOVA and Tukey’s test. In the Asian dataset, there were no significant differences between stages according to ERBB2 expression. (DOC 89 kb

Additional file 5: Table S3. of Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems

Stage comparison according to ERBB2 expression in Caucasian (GEO accession: GSE63288). For comparisons, we used one-way ANOVA and Tukey’s test. In the Caucasian dataset, there were significant stage differences according to ERBB2 expression. The significant p-value was in italic. (DOC 75 kb

BASCs express p110α and have Ser473-phosphorylated AKT.

<p>Immunofluorescent staining of tissue sections to detect triply stained (CCSP/SPC/p110α or pAKT) cells at terminal bronchi. BASCs encircled (x40 magnification). Calibration bar in lower right panel represents 50 μm.</p

BASC expansion at terminal bronchi of Pten^Δ5/Δ5; Kras^Lox/+; CCSP^Cre/+ mice.

<p>Immunofluorescent staining to detect cells at terminal bronchi that co-express CCSP and SPC. BASCs (encircled) illustrated at ×40 magnification. Bar graph indicates percentages of terminal bronchi with the indicated numbers of BASCS. Calibration bar in lower right panel represents 100 μm.</p

Kras^LA1 mice have an accumulation of BASCs at terminal bronchi.

<p>Immunofluorescent staining to detect cells at terminal bronchi that co-express CCSP and SPC. Encircled BASCs in top panels (x10 magnification) illustrated in lower panels at higher magnification (x40). Bar graph indicates percentages of terminal bronchi with the indicated numbers of BASCs. Calibration bars in images of H&E stained tissues represent 50 μm.</p

PX-866 treatment decreases BASC numbers in Kras^LA1 mice.

<p>Immunofluorescent staining to detect cells at terminal bronchi that co-express CCSP and SPC. Encircled BASCs in top panels (×10 magnification) illustrated in lower panels at higher magnification (×40). Bar graph indicates percentages of terminal bronchi with the indicated numbers of BASCS. Calibration bars in images of H&E stained tissues represent 50 μm.</p

Loss of PTEN expression in BASCs from Pten^Δ5/Δ5; Kras^Lox/+; CCSP^Cre/+ mice.

<p>Immunofluorescent staining of tissue sections to detect triply stained (CCSP/SPC/PTEN) cells at terminal bronchi. BASCs (encircled) illustrated at ×10 magnification. Calibration bar in lower right panel represents 100 μm.</p

PX-866 inhibits BASC expansion <i>in vitro.</i>

<p>(A) Sorted cells co-express SPC and CCSP. Kras^LA1 whole lung tissues were dissociated and sorted to isolate Sca-1^pos CD45^neg CD31^neg CD34^pos cells (P4 and P5 in left and middle panels, respectively), which were subjected to immunofluorescent staining (x40 magnification, right panel). Calibration bar in far right panel represents 10 μm. (B) BASCs form colonies when plated on feeder cultures. Photograph of colony (x10 magnification) formed upon initial plating (P1) and passage 2 (P2). Calibration bar in panel on right represents 50 μm. (C) BASCs differentiate when plated in matrigel. Cells were stained after 7 d in matrigel (x10 magnification) and the numbers of cells with features of alveolar type II cells (CCSP^neg SPC^pos), clara cells (CCSP^pos SPC^neg), or BASCs (CCSP^pos SPC^pos) were counted and expressed as the percentages of the total 158 counted cells in a pie chart. Examples of cells with features of ATII cells (ATII) and clara cells (CC) are indicated. Calibration bar in far right panel represents 50 μm. (D) PX-866 inhibits BASC colony formation. Photographs (x10 magnification) and quantification of colonies per well (5 wells per condition) formed after 6 d in the presence or absence of PX-866. Calibration bar in panel on right represents 50 μm.</p