Supplemental Figures and Legends

Supplemental Figures and Legends

Supplemental Figure Legends

Figure S1. HMGB1 promotes ATP production and subsequent tumor cell growth. Dose-response effects of HMGB1 on ATP production and cell growth in cultured human colon cancer (e.g. HCT116), leukemia (e.g. HL-60, Jurkat), and lung cancer cells (e.g. A549). Data represent relative ATP levels, with the untreated control set as 1 (mean ±SD, n = 3).

Figure S2. HMGB1, ATP, and CD11b-positive inflammatory cells are increased in human pancreatic tumors when compared with adjacent normal and control tissues. (A) H&E staining of pancreatic normal (“N”) or tumor (“T”) tissue sections. (B) Representative images of pancreatic normal (“N”) or tumor (“T”) (five different patients) tissue sections were analyzed by indirect immunofluorescence staining of HMGB1 (green), CD11b (red), and nuclear stain Hoechst 33342 (blue). (C) Quantification of HMGB1, CD11b, and ATP is shown relative to the normal control, which is set at 100 (mean ±SD, n = 5). ATP level in tissue was determined as described in the Materials and Methods section.

Figure S3. RAGE is found in the mitochondria obtained from mouse pancreatic cells and tissues. (A) Indirect immunofluorescence staining of RAGE (green), mitochondria (“Mit”, red) and nucleus (“Nuc”, blue) in RAGE wild-type (“WT”) and knockdown (“KD”) Panc02 cells and tissues in RAGE +/+ and RAGE -/- mice. The lower panel shows a higher magnification of pancreatic tissues with identified areas of RAGE staining (arrow); (B) Colocalization of RAGE and mitochondria from mouse pancreatea, heart, liver, and lung (mean ±SD) was quantified.

Figure S4. Overexpression of RAGE in mitochondria obtained from pancreatic tumor tissues. (A) Indirect immunofluorescence staining of RAGE (green), mitochondrial (“Mit”, red), and nuclear stain Hoechst 33342 (“Nuc”, blue) in pancreatic tumor (“T”) or nearby normal control (“N”) tissues. (B) Quantification of colocalized RAGE and mitochondria from normal and tumor pancreata (mean ±SD, n=3, *p0.001 versus normal group).

Figure S5. HMGB1 promotes RAGE expression in mitochondria. (A) Indirect immunofluorescence staining of RAGE (green), mitochondria (“Mit”, red) and nuclear stain Hoechst 33342 (“Nuc”, blue) in Panc02 cells treated with 10 μg/ml HMGB1 for 12-24 hours. (B) Quantification of colocalized RAGE and mitochondria is shown as mean ±SD (n = 3, *p < 0.001 versus control).

Figure S6. HMGB1 from necrotic cells is required to enhance RAGE expression in the mitochondria. (A) Panc02 cells were treated with 3×105 necrotic cells for 24 hours in the presence or absence of HMGB1 Ab or control IgG and then stained by indirect immunofluorescence for RAGE (green), mitochondria (“Mit”, red), and nuclear stains Hoechst 33342 (“Nuc”, blue). (B) Quantification of colocalized RAGE and mitochondria, NF-κB activity and cell migration. Data represent relative levels (mean ±SD, n = 3, *p< 0.001).

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