Fig 2: PQ association with the P584&Y586 residues of STIM1 promotes STIM1-TRPC1 interaction and increases intracellular calcium levels.a–b Co–IP analysis of STIM1-FLAG association with TRPC1-myc or TRPC1 D639A&D640A-myc (DDAA) (a) or TRPC1-myc association with STIM1-FLAG or STIM1 K778A&K779A-FLAG (KKAA) (b) in A549 cells treated w/wo 800 μM PQ for 30 min. c–e FACS analysis of intracellular calcium levels by Fluo-3 staining in A549 cells with ectopic expression of TRPC1 or DDAA (c), STIM1 or KKAA (d), or STIM1, STIM1 V585A&V611A (VVAA), STIM1 P584A&Y586A (PYAA), or STIM1 P584A&Y586A&G614A (PYGAAA) (e). Cells were treated w/wo 800 μM PQ for 18 hours. Mean ± s.d., *P < 0.05; ***P < 0.001; N.S., not significant. Two-Way ANOVA. n = 3. f Calcium imaging of A549 cells infected with STIM1, STIM1ΔK, KKAA, VVAA, PYAA, or PYGAAA. Cells were treated with 800 μM PQ, followed by the addition of 2 mM CaCl2. Mean ± sem. g Co–IP analysis of the TRPC1-myc association with STIM1-FLAG, PYAA-FLAG, or PYGAAA-FLAG in A549 cells treated w/wo 800 μM PQ for 30 min. h Pull-down analysis of PQ-Biotin association with STIM1 or PYAA in A549 cells. Cells were treated with 800 μM PQ-Biotin for 30 min. Immunoblot was probed using indicated antibodies.

Fig 3: Expression of STIM1 and Orai1 in rat spinal dorsal horn after RIPH.

Fig 4: Deficiency of STIM1 reduces autophagy in HCC. (A) After treatment with FCCP (20 μM, 6 h), the autophagy related proteins level of STIM1‐knockout (KO) cells and negative control group (NC) were detected by WB. The results were analyzed and normalized against respective expression in NC cells. (B) After treatment with FCCP (20 μM, 6 h), autophagosomes in NC and STIM1‐KO HepG2 cells, indicated by yellow arrows, were observed by transmission electron microscope (TEM). (C) mRFP‐GFP‐LC3 lentivirus was introduced into NC and STIM1‐KO HepG2 cells. After treatment with FCCP (20 μM, 6 h), yellow puncta represented the autophagosomes. Quantitative analysis of the yellow puncta. (D) After treatment with FCCP (20 μM, 6 h), the EMT proteins level of STIM1‐KO‐ and NC‐HepG2 or Hep3B cells were detected by WB. The results were analyzed and normalized against respective expression in NC cells. (E) The representative image of cancerous peripheral tissue in clinical HCC samples. N‐cadherin (purple), STIM1 (red), and LC3B (green) expression levels were detected by immunofluorescence (IF). The yellow line represents the edge of HCC tissue. The adjacent para‐cancerous tissue is at the bottom left corner (normal) and the HCC tissue (tumor) is at the top right corner. Blue represents DAPI. Data are expressed as mean ± SEM (n = 3). ***p < 0.001.

Fig 5: The effect of STIM1 C49A-C56A (2CA) or STIM2 C140A-C147A (2CA) mutation on their Ca2+-binding affinities in HeLa STIM1-STIM2 double knockout (HeLa SK) cells. Cartoons on the left are depictions of the types and locations of SC constructs used. (A) In-cell Ca2+ titration responses represented by FRET signals between co-expressed YFP-SOAR1L and PM localized STIM11-CC1 constructs, wild-type (WT) PM-SC1111-CFP, or PM-SC1111-2CA-CFP. Right, typical traces. (n = 3, more than 50 cells per measurement). (B) in situ Ca2+ titration curves shown by FRET signals between mNG△N5-SOAR1L and ER-localized STIM11-CC1 constructs, WT STIM11–310-CFP△C11, or STIM11–310-2CA-CFP△C11. Middle, typical FRET response; right, Ca2+ titration curves. (n = 3, more than 50 cells per measurement). (C) In-cell Ca2+ titration responses represented by FRET signals between co-expressed YFP-SOAR1L and PM localized STIM21-CC1 constructs, WT PM-SC2211-CFP or PM-SC2211-2CA-CFP. Right, typical traces. (n = 3, more than 50 cells per measurement). (D) in situ Ca2+ titration curves shown by FRET signals between mNG△N5-SOAR1L and ER-localized STIM21-CC1 constructs, WT SC2211-CFP△C11, or SC2211-2CA-CFP△C11. Middle left, typical RERT response; middle right, statistics of basal FRET efficiency; rightmost, Ca2+ titration curves. (n = 3, more than 50 cells per measurement, ****, p < 0.0001, Student’s t-test).