Supplementary Materials

Calumenin-15 Facilitates Filopodia Formation by Promoting TGF-b Superfamily Cytokine GDF-15 Transcription

Hui Feng1†, Liang Chen1†, Qiao Wang1†, Birong Shen1, Lin Liu1, Pengli Zheng1, Sizheng Xu1, Xiao Liu1, Jianguo Chen1, 2, Junlin Teng1*


Figure S1

Figure S1 Cloning of calumenin isoforms and signal peptides. (a) Primers designed for cloning calumenin isoforms. The forward primer (F-primer) and reverse primer (R-primer) are shown by the red arrows. (b) PCR products amplified by the primers in (a) from the cDNA of HeLa cells. The indicated bands were identified by sequencing. (c) Schematic picture shows the predicted signal peptide of calumenin-1 and calumenin-2 (Calu-1/2), calumenin-3 and calumenin-4 (Calu-3/4), which is marked by red box. (d) Representative pictures show the subcellular localizations of overexpressed EGFP fusion proteins (green) and GRIP1-mRFP (red). The signal peptides were fused with EGFP respectively. GRIP1-mRFP marks the Golgi apparatus. Bar, 10 µm.


Figure S2

Figure S2 Subcellular localization of calumenin-15-b-Gal-EGFP fusion protein (green). Nuclear DNA was stained with DAPI (blue). Bar, 10 µm. The upper panel shows the domain architecture of calumenin-15-b-Gal-EGFP.


Figure S3

Figure S3 Confirmation of the HeLa cell line stably expressing calumenin-15-EGFP or EGFP. Immunoprecipitation (IP) with antibody against EGFP were performed in the lysates of the HeLa cell line stably expressing calumenin-15-EGFP or EGFP, followed by immunoblotting (WB) with EGFP antibody.


Figure S4

Figure S4 Representative pictures of wound healing assay of the HeLa cells expressing EGFP or calumenin-15-EGFP. Images were acquired at 0, 12, 24, 29, 36, and 48 h post wounding. Bar, 100 µm.


Table S1 Information of primers used for quantitative real-time PCR.

F-RT-calumenin-15 / TGGGGGCTACGAGGAAAGATG
R-RT-calumenin-15 / TCCGCTCATC TCTAACCATCATCT
F-RT-GDF-15 / CTACAATCCCATGGTGCTCA
R-RT-GDF-15 / TATGCAGTGGCAGTCTTTGG
F-RT-ABHD2 / CACCTCTCTGAGCCTGTTCC
R-RT-ABHD2 / CGCAGATGTTCAGCAATGTT
F-RT-NELL2 / TGAAGGGAACCACCTACC
R-RT-NELL2 / ATTTGCCATCCACATACG
F-RT-ITPR1 / TGCCTCCACAATTCTACG
R-RT-ITPR1 / TGAATGTCCCACAGTTGC
F-RT-Myc / GAGGCGAACACACAACGTCTT
R-RT-Myc / CACGCAGGGCAAAAAAGCT
F-RT-Cyclin-D1 / GAACCTGGCCGCAATGAC
R-RT-Cyclin-D1 / CGCCTCTGGCATTTTGGA
F-RT-PML / GGAACGCGTTGTGGTGATC
R-RT-PML / TCGGGAGGACGAGTTTTCG
F-RT-Survivin / CTCAAGGACCACCGCATCTC
R-RT-Survivin / GCGCAGCCCTCCAAGA
F-RT-GAPDH / GGCATCCTGGGCTACACTGA
R-RT-GAPDH / GGAGTGGGTGTCGCTGTTG