HAP1 depletion in leukemic cells inhibits l-asparaginase–induced increase in [Ca²⁺]_i level. (A) l-Asparaginase–induced Ca²⁺ release from the ER is inhibited in HAP1-depleted cells. ER Ca²⁺ release in cells loaded with Fura-2-AM was measured by ratiometric single-cell Ca²⁺ imaging as described in “Methods and materials.” Graphs represent means of Ca²⁺ signal traces from 10 cells. Bar with 0 Ca²⁺ indicates the interval in which (Ca²⁺) in buffer is 0. (B) Analysis of the effects of HAP1 knockdown on ALL cell internal Ca²⁺ store capacity, and external Ca²⁺ entry and Ca²⁺ extrusion on treatment with l-asparaginase. [Ca²⁺]_i in cells loaded with Fura-2 were also performed on treatment with TBHQ with or without l-asparaginase or Ca²⁺ using single-cell Ca²⁺ imaging as described in “Methods and materials.” Values represent means of Ca²⁺ signal traces from 9 cells. (C-F) Response of control and HAP1-depleted cells were analyzed and compared. (C) Internal Ca²⁺ store capacity in HAP1-depleted cells is significantly reduced compared with control cells. Release of internal Ca²⁺ stores on treatment with TBHQ allows measurement of internal Ca²⁺ store capacity. In the presence of l-asparaginase, (D) Ca²⁺ entry is reduced, and (E) the rate of [Ca²⁺]_i influx is slower in HAP1-depleted cells compared with control cells. Comparison of the integrated Ca²⁺ signals (area under the curve from start of the Ca²⁺ signal until 22 minutes later) (F) showed decreased Ca²⁺ entry in cells depleted of HAP1, whereas the rate of extrusion of Ca²⁺ by the plasma membrane Ca²⁺ pumps, observed as the decline in [Ca²⁺]_i following removal of external Ca²⁺, appears to be similar. (G) HAP1-depleted cells have reduced resting [Ca²⁺]_i level compared with control cells. Resting [Ca²⁺]_i levels were measured as described in “Methods and materials.” Values in C-G are means ± standard error of the mean from 3 independent experiments. **P < .05.