JCS041640 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effects of genistein and U-73122 on BCR-mediated Ca²⁺ responses. (A) WT DT40 cells were stimulated with 2 µg/ml anti-IgM in nominally Ca²⁺-free medium. (B,C) WT DT40 cells were pretreated with 50 µM genistein (B) or 2 µM U-73122 (C), followed by the addition of 2 µg/ml anti-IgM in nominally Ca²⁺-free medium. Traces shown are the means ± s.e.m. of normalized fluorescence ratios. The numbers of control, genistein-treated and U-73122-treated cells were 162, 179 and 175, respectively. Horizontal bars indicate the presence of anti-IgM, U-73122, and genistein. Time points for the addition of reagents are indicated by the arrows.

• Supplemental Figure S2 -

  Fig. S2. Effects of trypsin on Ca²⁺ entry in ThG-pretreated cells. (A) Trypsin-induced Ca²⁺ responses in WT DT40 cells. WT DT40 cells were stimulated with 100 U/ml trypsin in nominally Ca²⁺-free medium. Trace shown is the mean ± s.e.m. of all cells examined (197 cells). Horizontal bars indicate the presence of trypsin. Trypsin was added at the time indicated by the arrow. (B) WT (black) and IP3R-KO (red) DT40 cells were treated with ThG in nominally Ca²⁺-free medium, followed by the addition of 100 U/ml trypsin in the presence of La³⁺ and Ca²⁺. Traces shown are the means ± s.e.m. of 151 and 160 WT and IP3R-KO cells, respectively. The horizontal bars indicate the presence of ThG, trypsin, La³⁺ and Ca²⁺. Time points for the addition of reagents by exchanging medium are indicated by the arrows.

• Supplemental Figure S3 -

  Fig. S3. Ca²⁺ entry in the presence of 1 µM La³⁺ or 1 µM Gd³⁺. YFP-STIM1-overexpressing Stim1-KO cells were treated with ThG in nominally Ca²⁺-free medium, followed by the addition of 2 µg/ml anti-IgM in the presence of Ca²⁺ and 1 µM La³⁺ (A,B) or 1 µM Gd³⁺ (C). (A) Mean ± s.e.m. of normalized fluorescence ratios in the presence of 1 µM La³⁺ (14 cells). (B,C) Typical Ca²⁺ responses in single YFP-STIM1-overexpressing Stim1-KO cells, in the presence of 1 µM La³⁺ (B) or 1 µM Gd³⁺ (C). Horizontal bars indicate the presence of ThG, anti-IgM, La³⁺, Gd³⁺, and Ca²⁺. Time points for the addition of reagents are indicated by the arrows.

• Supplemental Figure S4 -

  Fig. S4. Effects of 2-APB and SKF96365 on SOC and B-SOC. (A) WT cells were treated with 1 µM ThG in nominally Ca²⁺-free medium, followed by the addition of Ca²⁺ in the absence (black) or presence (red) of 2-APB, or in the presence of SKF96365 (blue). Traces shown are the means ± s.e.m. of 216 untreated cells, and 202 or 200 cells treated with 2-APB or SKF96365, respectively. Horizontal bars indicate the presence of ThG and Ca²⁺. Time points for the addition of reagents by exchanging media are indicated by the arrows. The red and blue arrows indicate the time of 2-APB or SKF96365 addition, respectively. (B) WT DT40 cells were treated with ThG in nominally Ca²⁺-free medium. Subsequently, cells were treated with 75 µM 2-APB (red), or 10 µM SKF96365 (blue) or without any inhibitors (black), followed by addition of La³⁺ and Ca²⁺ and stimulation with anti-IgM. Traces shown are the means ± s.e.m. of 209 untreated cells, and 190 and 205 cells treated with 2-APB or SKF96365, respectively. The horizontal bars indicate the presence of ThG, La³⁺, Ca²⁺, and anti-IgM. Time points for the addition of reagents by exchanging media are indicated by the arrows. The red and blue arrows indicate the time of 2-APB or SKF96365 addition, respectively. (C) Responses in the absence (black) or presence of 2-APB (red) or SKF96365 (blue) from the boxed region in B are shown. The horizontal bars indicate the presence of anti-IgM, La³⁺, and Ca²⁺. Arrows indicate the times of solution exchange.

• Supplemental Figure S5 -

  Fig. S5. La³⁺-resistant Ca²⁺ entry in Jurkat T cells. (A) Jurkat T cells were stimulated with 3 µg/ml anti-CD3 in nominally Ca²⁺-free medium, followed by the addition of 1.3 mM Ca²⁺ in the absence (black) or presence (red) of 1.0 µM La³⁺. Traces shown are the means ± s.e.m. of all cells examined (167 and 179 cells in the absence and presence of La³⁺, respectively). Horizontal bars indicate the presence of anti-CD3 and Ca²⁺. Time points for the addition of reagents by exchanging media are indicated by the arrows. The red arrow indicates the time of La³⁺ addition. (B) Jurkat cells were treated with 1 µM ThG in nominally Ca²⁺-free medium, followed by the addition of Ca²⁺ (black) or followed by the addition of La³⁺, Ca²⁺, and 3 µg/ml anti-CD3 (red). Traces shown are the means ± s.e.m. of 128 cells in the absence of La³⁺ and 162 cells in the presence of La³⁺ and anti-CD3. The horizontal bars indicate the presence of ThG, Ca²⁺, and anti-CD3. Time points for the addition of reagents by exchanging media are indicated by the arrows. The red arrow indicates the time of La³⁺ addition.