Mab305

In IHC, paraffin blocks of the xenograft tumors were cut into 4-μm sections. The slides were incubated with primary antibodies: IGFBP3 (1:200, MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:500, H1alpha67-ChIP Grade; Abcam, Cambridge, UK), HIF-2α (1:1,000, ab8365; Abcam, Cambridge, UK), HO-1 (1:200, GTX101147, Genetex, Irvine, CA, USA), or Von Hippel-Lindau (VHL) (1:200, GTX101087, Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
In ICC, 0.2 × 10⁵ cells were cultured on 25 mm × 25 mm glass slides and incubated under hypoxic or normoxic conditions for 17 h. Then, the slides were incubated with primary antibodies: IGFBP3 (1:200; MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:300; GTX127309; Genetex, Irvine, CA, USA), or HIF-2α (1:300; GTX30114; Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
A chromogen in IHC and ICC was developed, following the protocol of UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, Waltham, MA, USA). The sections or slides were stained with hematoxylin for examination, and photos were taken using Moticam X3 Plus (Motic, Speed Fair Co., Ltd, HK) microscope camera by Motic Images Plus 3.0 software. The signals of IHC and ICC were quantified and analyzed by ImageJ 1.53 k software (NIH, USA).

Total cell proteins were prepared from the DM1 model cells, as previously described (Nakamori et al., 2017 (link)). Then, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the following primary antibodies: mouse anti-IGFBP3 (1:500; MAB305, R&D Systems), rabbit anti-PAI-1 (1:2000; NBP1-19773, Novus), rabbit anti-AKT (1:500; GTX121937, GeneTex), rabbit anti-phospho-Akt (Ser473) (1:500; 4,058, Cell Signaling Technology), rabbit anti-p53 (1:100; 2,527, Cell Signaling Technology), rabbit anti-p21 (1:1,000; 2,947, Cell Signaling Technology), rabbit anti-p16 (1:1,000; ab108349, Abcam), and rabbit anti-GAPDH (1:1,000; G9545, Sigma-Aldrich). After incubation, the immunoblots were washed, incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin (Ig) G or anti-rabbit IgG (GE Healthcare), and detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare) using a ChemiDoc Touch Imaging System (Bio-Rad).

The promoter fragments of IGFBP3 (+ 160/ − 1414, + 160, 5′-AAACTCGAGGCATTCGTGTGTACCTCGTG-3′;–1414, 5′-CCCGAGCTCTGATCTTCCCCTGTCCACTC-3′), HIF-1a (+ 80/ − 1871, + 80, 5′- AAACTCGAGCTCTCCTCAGGTGGCTTGTC-3′;–1871, 5′- CCCGAGCTCGAGTTGCAGTGAGCCGAAAT-3′), and HIF-2a (+ 46/-1222, + 46, 5′-AAACTCGAGGAGGACAAGCTGGCAGAGAC-3′;–1222, 5′- CCCGAGCTCGTGTTCCGCATTTTGGAAGT-3′) were generated by chromosomal DNA extraction from P0, amplified by Q5 High-Fidelity DNA Polymerase ((NEB, Ipswich, MA, USA), and constructed into pGL3 (Promega, Madison, WI, USA). The pGL3-IGFBP3, pGL3-HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 10⁴ cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA). Luciferase activities were calculated relative to the empty pGL3 transfectants.