American Society of Hematology

Figure 3.

Clearance, STIM1 protein levels and Ca2+-signaling in WT and KI-STIM1I115F platelets. (A) Platelet lifespan in the indicated groups (n = 6). Mann-Whitney U test (n = 6 per group) P value: ^^^≤0.0015 and ^^≤0.0028 for WT vehicle vs KI-STIM1I115F vehicle; ##≤0.003 and #≤0.0014 for KI-STIM1I115F vehicle vs KI-STIM1I115F CIC-39Na. (B) Representative images and densitometry analysis of western blots for STIM1 and ORAI1 (n = 3); unpaired t test for STIM1 blot (P value: ****<0.0001 WT vehicle vs WT CIC-39Na; ###<0.0002 KI-STIM1I115F vehicle vs KI-STIM1I115F CIC-39Na and ^^^0.009 WT vehicle vs KI-STIM1I115F vehicle). ORAI1 blot (P value: ****<0.0001 WT vehicle vs WT CIC-39Na; ####<0.0001 KI-STIM1I115F vehicle vs KI-STIM1I115F CIC-39Na). (C) Basal platelet cytosolic Ca2+. Values are median ± SEM of at least 300 platelets from 2 experimental days. Mann-Whitney U test (n = 6 per group) P value: ^^^^<0.0001 WT vehicle vs KI-STIM1I115F vehicle and ####<0.0001 KI-STIM1I115F vehicle vs KI-STIM1I115F CIC-39Na. (D) Average Ca2+-traces depicting tBHQ release and SOCE. Statistical analysis of the AUC of the first 200 seconds after the respective addition are present in the insets. Arrows indicate addition of the SERCA poison in Ca2+-free conditions and the readdition of Ca2+. Traces are average of 300 cells from 2 different experimental days and color coding is as in panel C; to allow comparisons, F/Fo was offset to 0 in the graphs for all traces. Mann-Whitney U test. P value: ^^^^<0.0001 for WT vehicle vs KI-STIM1I115F vehicle; ###<0.001 for KI-STIM1I115F vehicle vs KI-STIM1I115F CIC-39Na. (E-F) Average Ca2+traces induced by the indicated agonists in Ca2+-containing media. Traces are average of 300 cells from 2 different experimental days; statistical analysis of the AUC of the first 200 seconds after the respective addition are present in the insets and color coding is as in panel C; Mann-Whitney U test. P value: ****<0.0001 and **<0.003 for KI-STIM1I115F vehicle vs KI-STIM1I115F CIC-39Na; ^^^<0.001 for WT vehicle vs KI-STIM1I115F vehicle. To allow visual comparisons, F/Fo was offset to 0 in the graphs for all traces.

Clearance, STIM1 protein levels and Ca²⁺-signaling in WT and KI-STIM1^I115F platelets. (A) Platelet lifespan in the indicated groups (n = 6). Mann-Whitney U test (n = 6 per group) P value: ^^^≤0.0015 and ^^≤0.0028 for WT vehicle vs KI-STIM1^I115F vehicle; ##≤0.003 and #≤0.0014 for KI-STIM1^I115F vehicle vs KI-STIM1^I115F CIC-39Na. (B) Representative images and densitometry analysis of western blots for STIM1 and ORAI1 (n = 3); unpaired t test for STIM1 blot (P value: ****<0.0001 WT vehicle vs WT CIC-39Na; ###<0.0002 KI-STIM1^I115F vehicle vs KI-STIM1^I115F CIC-39Na and ^^^0.009 WT vehicle vs KI-STIM1^I115F vehicle). ORAI1 blot (P value: ****<0.0001 WT vehicle vs WT CIC-39Na; ####<0.0001 KI-STIM1^I115F vehicle vs KI-STIM1^I115F CIC-39Na). (C) Basal platelet cytosolic Ca²⁺. Values are median ± SEM of at least 300 platelets from 2 experimental days. Mann-Whitney U test (n = 6 per group) P value: ^^^^<0.0001 WT vehicle vs KI-STIM1^I115F vehicle and ####<0.0001 KI-STIM1^I115F vehicle vs KI-STIM1^I115F CIC-39Na. (D) Average Ca²⁺-traces depicting tBHQ release and SOCE. Statistical analysis of the AUC of the first 200 seconds after the respective addition are present in the insets. Arrows indicate addition of the SERCA poison in Ca²⁺-free conditions and the readdition of Ca²⁺. Traces are average of 300 cells from 2 different experimental days and color coding is as in panel C; to allow comparisons, F/Fo was offset to 0 in the graphs for all traces. Mann-Whitney U test. P value: ^^^^<0.0001 for WT vehicle vs KI-STIM1^I115F vehicle; ###<0.001 for KI-STIM1^I115F vehicle vs KI-STIM1^I115F CIC-39Na. (E-F) Average Ca²⁺traces induced by the indicated agonists in Ca²⁺-containing media. Traces are average of 300 cells from 2 different experimental days; statistical analysis of the AUC of the first 200 seconds after the respective addition are present in the insets and color coding is as in panel C; Mann-Whitney U test. P value: ****<0.0001 and **<0.003 for KI-STIM1^I115F vehicle vs KI-STIM1^I115F CIC-39Na; ^^^<0.001 for WT vehicle vs KI-STIM1^I115F vehicle. To allow visual comparisons, F/Fo was offset to 0 in the graphs for all traces.

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