Physiological role of TPC2 on osteoclast

SUPPLEMENTAL DATA

Lysosomal cCalcium channel, TPC2, regulates osteoclastogenesis via generation of intracellular calcium response and subsequent NFATc1 localization

Takuya Notomi1, 2, Yoichi Ezura1, and Masaki Noda1,2

1Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, 113-8510, Tokyo, Japan

2Global Center of Excellence Program for Molecular Science for Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan

Correspondence: Masaki Noda, Tel & FAX: +81-3-5803-4061, E-mail:

Co-correspondence: Takuya Notomi, Tel & FAX: +81-3-5803-4061,E-mail:

Running title: Physiological role of TPC2 on osteoclast

SUPPLEMENTAL FIGURE LEGENDS

Figure S1, Knockdown efficiency of TPC2 expression level in osteoclast precursor cells.

A and B, TPC2 mRNA levelin BMMand RAW cells transfected with lentivirus-based miR-RNA. The mRNA expression levels of TPC2 were analyzed based on QT-PCR in BMM (A) and RAW (B) cell lines.C, TRAP-staining of osteoclasts developed in the culture of RAW cells on day 3 of RANKL treatment. Scalebar=100μm.

Figure S2, The changes of [Ca2+]i are regulated by TPC2 in osteoclast precursor cells.

A, TPC2 related [Ca2+]i in mono-nucleated cells of RAW (Day3) ; Peak analysis (peak curve area) histograms obtained from each 15 experiments. Total cell number is 361 (Control) and 323 (Knockdown B). Threshold was decided by fitted polynomial curve (Control; black curve line). The number shows cell number and percentage of “No responding cell” or “Responding cell” (Control vs. Knockdown, p<0.0001; chi-square test). B, TPC2 related [Ca2+]i in mono-nucleated cells of BMM (Day3). Peak analysis (peak curve area) histograms obtained from each 4 experiments. Total cell number is 79 (Control) and 67 (Knockdown). The number shows cell number and percentage of “No responding cell” or “Responding cell” (Control vs. Knockdown, p<0.0001; chi-square test).C, The representative Ca2+ plots in control- and TPC2-KD RAW cells(1 plot per 1 cell) (representative 20 plots out of 361 (control) and 323 (TPC2-KD) cells). Each line represents individual single cells. The number shows percentage of “No responding cell” or “Responding cell”.D, The percentage of responding cells in RAW cell lines with/without RANKL. * p<0.01 (n=10-15).

Figure S3, Bafilomycin-sensitive [Ca2+]i exists independent of thapsigargin-sensitive ER-Ca2+ store.

A, Lysosome related [Ca2+]i in mono-nucleated cells of RAW (Day3) ; Peak analysis histograms obtained from 20-25 experiments. Total cell number is 450 (Tg), 410 (Tg-Baf) and 150 (Baf). The histograms were fitted with Gaussian curve (excluding Control). The arrow in the Tg-Baf panel shows that Baf-treatment shift the Gaussian curve to the left (Red curve line: Tg-Baf, Green: Tg). B, The average of integrated Ca2+ peak curve area in Tg, Tg-Baf and Baf treated RAW cells. (n=450, 410 and 150). * p<0.01.

Physiological role of TPC2 on osteoclast