Fig 1: Immunohistochemistry confirms expression of LIF, C1QA, and TREM2 in BE and EAC tissues. Immunohistochemical staining pattern of (A) LIF, (B) C1QA, and (C) TREM2 in normal squamous (SQ), BE, and EAC tissues showing glandular membranous and perinuclear expression in epithelial cells in addition to expression by infiltrating or resident immune cell types. Images are from digitally zoomed fields of view from scanned slides using ImageScope (Leica). Original magnification: top, 10×; bottom, 20×.
Fig 2: LIF, C1QA, and TREM2 mRNA expression in the EAC cancer sequence. (A) Hierarchical gene and condition clustering of siRNA gene targets (Z-score < –1.645 in siRNA library screening data) and tissues (EAC and squamous control tissues [SQ]) respectively, in gene expression microarray data (P < .001 in moderated t test and FC > 1.5) as previously reported.25Blue and red dendrites of conditional clustering signify EAC and SQ tissues respectively. Heat map yellow and blue coloring denote low and high expression levels respectively. In hierarchical clustering GEM data yellow and blue coloring reflects low and high expression respectively. (B) Top 10 genes overexpressed in EAC tissues ranked by the strength of effect achieved in siRNA screening data (robust Z score). Real-time reverse-transcription polymerase chain reaction analysis of (C) LIF, (D) C1QA, and (E) TREM2 mRNA expression in normal esophageal (SQ), BE, and EAC tissues. ****P < .0001 in Mann–Whitney testing for differences in mRNA tissue expression. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Fig 3: C1q is produced by EAC cells and promotes cell proliferation in an autocrine manner. (A) Expression of C1QA protein in EAC cell lines by Western blot. (B) Silencing of C1QA results in reduced EAC cell viability with no effects on HEK-293 cells. (C) C1QA protein expression by Western blot after siRNA-mediated silencing or treatment with C1 inhibitor protein C1INH in SKGT4 EAC cells. (D) Densitometry of panel C. (E) Cell viability and (F) C1QA mRNA levels in SKGT4 EAC cells after either silencing of C1QA or treatment of siC1QA-silenced cells with native C1q to examine recovery of proliferative phenotypes. (G) Cell viability of multiple EAC cell lines after siRNA-mediated silencing of TREM2. (H) Treatment of TREM2-silenced SKGT4 EAC cells with rLIF or C1q native protein resulted in the rescue of the observed growth arrest. (I) Expression of TREM2 and C1q-associated ITAM docking protein TYROBP (DAP12), signaling intermediary SYK and p-SYK in multiple EAC cell lines. **P < .01, ***P < .001, and ****P < .0001 in either Student t test (proliferation) or Mann–Whitney testing (mRNA expression). siC1QA, siRNA pools targeting complement C1q subcomponent subunit A; siNT, nontargeting siRNA pool; siTREM2, siRNA pools targeting triggering receptor expressed on myeloid cells 2.
Fig 4: LIF hierarchically regulates STAT3 phosphorylation, IL6 expression, and cell proliferation in EAC cells. SiRNA-mediated silencing of LIF expression results in reduced LIF mRNA (A) expression, (B) secretion, and (C) a recombinant-recoverable reduction in cell proliferation in CP-D, SKGT4, and OE-33 EAC cell lines. Treatment of LIF silenced cells with recombinant LIF protein (rLIF) results in (D) recovery of LIF mRNA in CP-D, SKGT4, and OE33 EAC cells and (E) protein expression levels in SKGT4 EAC cells. (F) STAT3-Y705 phosphorylation levels in LIF-silenced SKGT4 EAC cells are rescued by treatment with recombinant LIF protein as determined by Western blot. (G) Silencing LIF expression in SKGT4 EAC cells results in decreased IL6 mRNA expression that is recovered upon exposure to rLIF protein. (H) Neither silencing IL6 expression by siRNA-mediated silencing nor treatment with anti-IL6 antibody affects LIF mRNA levels or SKGT4 EAC cell growth. (I) Silencing LIF expression results in decreased expression of C1QA mRNA but not TREM2 in SKGT4 EAC cells. ∗P < .05, **P < .01, ***P < .001, and ****P < .0001 in either Student t test (proliferation) or Mann–Whitney testing (mRNA expression). siNT, nontargeting siRNA pool; siLIF, LIF-targeted siRNA pool; rLIF, recombinant LIF protein; rIL6, recombinant IL6 protein.
Supplier Page from Abcam for Anti-C1QA antibody [EPR2979(2)]