Journal: Biomaterials Research

Article Title: Mesenchymal Stem Cell-Derived Exosomes Inhibit Stim1–Orai1 Signaling and Calcium Overload-Induced Mitochondrial Damage of Follicular Helper T Cells in Lupus

doi: 10.34133/bmr.0255

Figure Lengend Snippet: MSC-Exos regulate calcium homeostasis in Tfh. (A) Volcano diagram of DEGs of Tfh between control group and Exo group ( n = 3). (B) KEGG enrichment analysis of Tfh between control group ( n = 3) and Exo group ( n = 3). These pathways were significant differences in the diagram. (C) GO enrichment analysis of DEGs between Tfh and naïve CD4 + T cells in C57 based on the GSE157648 dataset. (D) Fluo-4 AM-loaded Th0 and Tfh treated with/without MSC-Exos in HBSS medium for 30 min, and intracellular Ca 2+ levels were detected by flow cytometry using Fluo-4 AM as the probe ( n = 5). (E) mRNA expression levels of Stim1, Stim2, and Orai1 in Tfh with PBS or MSC-Exos coculture were detected by RT-qPCR. (F) WB analysis of Stim1 and Orai1 protein expression in Th0 and Tfh after treatment with MSC-Exos or PBS. (G) WB analysis of Stim1 and Orai1 protein expression in Tfh treated with/without MSC-Exos, and ionomycin (5 μM) was used to elevate intracellular Ca 2+ levels. (H) RT-qPCR analysis of NFATc1, NFATc2, and CaN mRNA expression levels in Tfh after treatment with MSC-Exos or PBS. (I) WB analysis of CaN and NFATc2 protein expression in Tfh treated with/without MSC-Exos. (J) WB analysis of IκB, p-IκB, P65, and p-P65 protein expression in Tfh treated with/without MSC-Exos. Bars indicate the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 determined by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: These membranes were blocked with 5% nonfat milk at 25 °C for 1 h. Primary antibodies targeting CaN (Proteintech, 13422-1-AP, China), NFATc2 (Proteintech, 22023-1-AP, China), P65 (Affinity, AF5006, USA), p-P65 (Affinity, AF2006, USA), IκB (Abmart, T55026 , China), p-IκB (Abmart, TA2002, China), Stim1 (Proteintech, 11565-1-AP, China), Orai1 (Proteintech, 28411-1-AP, China), and MCU (Abways, BY0101, China) were incubated overnight at 4 °C.

Techniques: Control, Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Biomaterials Research

Article Title: Mesenchymal Stem Cell-Derived Exosomes Inhibit Stim1–Orai1 Signaling and Calcium Overload-Induced Mitochondrial Damage of Follicular Helper T Cells in Lupus

doi: 10.34133/bmr.0255

Figure Lengend Snippet: MSC-Exos regulate Tfh differentiation and function via a multi-target network, restoring the Tfh/Tfr balance and alleviating SLE. Mechanistically, MSC-Exos inhibit Tfh activation by correcting intracellular calcium dysregulation and preventing mitochondrial calcium overload. In the cytoplasm, MSC-Exos restrict calcium influx through inhibition of the Stim1/Orai1 expression, thereby reducing NFAT and NF-κB activation. In the mitochondria, MSC-Exos suppress aberrant MCU expression, mitigating calcium overload-induced mitochondrial damage, and restoring mitochondrial homeostasis in Tfh. Figure was created with BioGDP .

Article Snippet: These membranes were blocked with 5% nonfat milk at 25 °C for 1 h. Primary antibodies targeting CaN (Proteintech, 13422-1-AP, China), NFATc2 (Proteintech, 22023-1-AP, China), P65 (Affinity, AF5006, USA), p-P65 (Affinity, AF2006, USA), IκB (Abmart, T55026 , China), p-IκB (Abmart, TA2002, China), Stim1 (Proteintech, 11565-1-AP, China), Orai1 (Proteintech, 28411-1-AP, China), and MCU (Abways, BY0101, China) were incubated overnight at 4 °C.

Techniques: Activation Assay, Inhibition, Expressing

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: Additional data related to . (A and B) Expression of STIM1 and STIM2 in MDA-MB-231 (A) and LM2-4 (B) cells following CRISPR/Cas9 gene editing. Representative images from WB analysis of lysates prepared from the indicated MDA-MB-231 (A) or LM2-4 (B) cells using antibodies for STIM1, STIM2, actin, GAPDH, or tubulin. Clones F10 and E5 are shown here as additional controls and were not used in the present study. (C) Representative images (left) and quantitation (right) of adhesion assays ( n = 5) using the indicated MDA-MB-231 cells. The field of view in each image measures 2 × 2 mm. (D and E) Representative images (left) and quantitation (right) of two invasion assays using WT, S1KO, S2KO, dKO (C4), or dKO(B6) MDA-MB-231 cells (D) or using dKO(B6) cells re-expressing EYFP-STIM1 or EYFP-STIM2 (E). Scale bar = 300 μm. Results from each experiment in C or in D were normalized to WT cells, while data from E were normalized to control dKO cells. Bars show the mean ± SEM. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Expressing, CRISPR, Clone Assay, Quantitation Assay, Control

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: Deletion of both STIM isoforms rescues cell migration. (A) Representative images of WT, S1KO, S2KO, or dKO migrating cells, as indicated. (B) Scale bar = 350 μm (B)Number of migrating cells from each group was normalized to that of control (WT) cells. Bars show the mean ± SEM (WT, n = 18; S1KO, n = 13; S2KO, n = 15; dKO-C4, n = 8; dKO-B6, n = 7). (C and D) EYFP-STIM1 or EYFP-STIM2 was re-expressed in dKO cells. (C) Representative images of migrating cells from the indicated cells. Scale bar = 350 μm. (D) Number of migrating cells from STIM1 or STIM2 rescue cells was normalized to that of control (dKO) cells. Bars show the mean ± SEM (control, n = 7; STIM1, n = 6; STIM2, n = 7). (E and F) WB analysis of lysates from WT, S1KO, S2KO, and dKO cells using two different antibodies against pMLC (ab2480 or cs3675) and GAPDH as a loading control. (E) Representative images from two independent experiments. (F) Quantification of pMLC signal intensity relative to that of GAPDH and normalized to control (WT) cells. (G and H) Bars shows the mean ± SEM of normalized pMLC ( n = 4) (G and H). Representative confocal images (G) and quantification (H) of anti-phosphorylated MLC (red) and phalloidin (F-actin, green) fluorescence (see the Materials and methods section). Bars show the mean ± SEM of pMLC staining at actin bundles per cell in the indicated cell type (WT, n = 30; S1KO, n = 28; S2KO, n = 32; dKO, n = 30). (I and J) Representative images (I) and quantification (J) of anti-paxillin (red) and phalloidin (F-actin, green) fluorescence (see the Materials and methods section). Bars show the mean ± SEM of FAs per cell in the indicated cell type (WT, n = 30; S1KO, n = 34; S2KO, n = 31; dKO, n = 36). Scale bar = 10 μm. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Migration, Control, Fluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: Additional data related to , and . (A) Cells were cultured on Matrigel-coated polyacrylamide gels of the indicated stiffness or on glass. The average intracellular Ca 2+ responses in Fura-2–loaded cells following the application of a Ca 2+ -free solution containing 200 µM ATP are shown for each condition ( n = 202 for both WT and dKO cells). (B–D) Mean rise and decay times of Ca 2+ puff fluorescence were measured during increases and decreases to 20%, 50%, 80%, and 100%, analyzed from the indicated type of MDA-MB-231 (B) or LM2-4 (C) cells shown in or from MDA-MB-231 STIM dKO control or STIM1, STIM2, or STIM1 D76A–expressing (rescue) cells (D) shown in . (E) Quantification of resting Ca 2+ levels in the indicated cells following loading with a Ca 2+ indicator (Fura-2), caged IP3 (ci-IP3/PM), and EGTA-AM. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05.

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Cell Culture, Fluorescence, Control, Expressing

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: MDA-MB-231 STIM1 KO (S1KO). Time-lapse TIRFM of S1KO MDA-MB-231 cells co-loaded with Cal-520 AM, EGTA-AM, and caged-IP3. IP3 was uncaged at 10 s (frame 500) using a 405-nm laser pulse. Images were acquired at 50 frames per second. This video relates to .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques:

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: MDA-MB-231 STIM1/STIM2 dKO. Time-lapse TIRFM of dKO MDA-MB-231 cells co-loaded with Cal-520 AM, EGTA-AM, and caged-IP3. IP3 was uncaged at 10 s (frame 500) using a 405-nm laser pulse. Images were acquired at 50 frames per second. This video relates to .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques:

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: Analysis of STIM expression and function in LM2-4 cells. (A) Average intracellular Ca 2+ responses during SOCE are shown for WT ( n = 137), S1KO ( n = 70), S2KO ( n = 73), and S1/S2 dKO ( n = 87) LM2-4 cells. (B and C) Quantification of SOCE (B) and Tg-induced ER Ca 2+ release (C) for individual cells depicted in A. The AUC was calculated from 400 to 1,200 s. (D) Analysis of SOCE in Fluo-4–loaded LM2-4 cells ( n = 46) compared with MDA-MB-231 cells ( n = 47). (E and F) Representative images (left) and quantification (right) of WB analysis ( n = 3–4) of lysates from LM2-4 and MDA-MB-231 cells using antibodies against STIM1, STIM2, or actin, as indicated. (G) Traces of cell-wide Ca 2+ dynamics recorded from individual WT, S1KO, S2KO, and dKO LM2-4 cells using the experimental protocol described in . Upper inset shows a pie chart of the fraction of cells showing global Ca 2+ rise (blue) following UV-induced uncaging of IP3. (H) Total cell lysates were prepared from S1/S2 dKO HEK293 cells expressing either EYFP-STIM1 WT, the D76A mutant, or EGFP. WB images of the IP protein material and eluted fractions are shown, demonstrating the absence of interaction between STIM1 and IP3R2 or IP3R3. Bars show the mean ± SEM. Statistics: one-way ANOVA with Tukey’s post hoc test (B and C) or two-tailed t test (F); *P < 0.05, **P < 0.01, ***P < 0.001. AUC, area under the curve; IP, immunoprecipitated. Source data are available for this figure: .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Expressing, Mutagenesis, Two Tailed Test, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: LM2-4 STIM1 KO (S1KO). Time-lapse TIRFM of S1KO LM2-4 cells co-loaded with Cal-520 AM, EGTA-AM, and caged-IP3. IP3 was uncaged at 10 s (frame 500) using a 405-nm laser pulse. Images were acquired at 50 frames per second. This video relates to .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques:

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: LM2-4 STIM1/STIM2 dKO. Time-lapse TIRFM of dKO LM2-4 cells co-loaded with Cal-520 AM, EGTA-AM, and caged-IP3. IP3 was uncaged at 10 s (frame 500) using a 405-nm laser pulse. Images were acquired at 50 frames per second. This video relates to .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques:

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: Restoring STIM1 or STIM2 expression rescues IP3-dependent Ca 2+ release through a diffuse release mode. (A) Proposed model for STIM regulation of diffuse Ca 2+ release via IP3R. Under ER Ca 2+ replete conditions, STIM is at a resting conformation and IP3R-mediated Ca 2+ release occurs primarily via local Ca 2+ puffs. Upon a decrease in [Ca 2+ ] ER , STIM adopts an active conformation and promotes a diffuse mode of Ca 2+ release via IP3R. (B) Average (mean ± SEM) intracellular Ca 2+ responses of basal and SOCE are shown for control dKO ( n = 67) or for dKO cell expressing STIM1 ( n = 85), STIM2 ( n = 89), or STIM1 D76A ( n = 58) cells. (C) Boxplot shows quantification of Tg-induced ER Ca 2+ release for individual cells shown in B. (D–J) Analysis of global and puff Ca 2+ release in MDA-MB-231 control dKO cells ( n = 13) or in cells expressing EYFP-STIM1 ( n = 19), EYFP-STIM2 ( n = 23), or EYFP-STIM1 D76A ( n = 29). (D) Representative traces of Cal-590 fluorescence ratios (ΔF/F0) recorded from the center of an individual puff site before and after ci-IP3 uncaging by a pulse (1 s) of 405-nm light in the indicated cells. (E and F) Boxplot shows the number of Ca 2+ puffs (E) or puff sites (F) per cell for the indicated cells. (G) Average global Ca 2+ responses recorded after UV-induced IP3 uncaging for the indicated cells. (H) Time course of Ca 2+ release via Ca 2+ puff in the indicated cells was quantified as in . Note that in cells expressing STIM1 or STIM2, a rise in global Ca 2+ levels (G) is accompanied by a decrease in localized Ca 2+ release (H). However, in cells expressing the STIM1 D76A mutant, the global Ca 2+ increase occurs without a corresponding decrease in localized release. (I) Amplitude distributions of local Ca 2+ puffs in the indicated cells. (J) Curves show the normalized Gaussian fit to the data shown in I for the indicated type of cells. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05, ***P < 0.001.

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Expressing, Control, Fluorescence, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: MDA-MB-231 dKO + EYFP-STIM1. Time-lapse TIRFM of dKO MDA-MB-231 cells expressing EYFP-STIM1 and co-loaded with Cal-520 AM, EGTA-AM, and caged-IP3. IP3 was uncaged at 10 s (frame 500) using a 405-nm laser pulse. Images were acquired at 50 frames per second. This video relates to .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: MDA-MB-231 dKO + EYFP-STIM1 D76A. Time-lapse TIRFM of dKO MDA-MB-231 cells expressing EYFP-STIM1 D76A and co-loaded with Cal-520 AM, EGTA-AM, and caged-IP3. IP3 was uncaged at 10 s (frame 500) using a 405-nm laser pulse. Images were acquired at 50 frames per second. This video relates to .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: STIM-IP3R crosstalk regulates migration of breast cancer cells

doi: 10.1083/jcb.202411203

Figure Lengend Snippet: Mechanistic model of STIM-IP3R crosstalk in regulating breast cancer cell migration. Breast cancer cell migration is regulated by a dynamic interplay between STIM proteins and IP3Rs, which together shape the spatiotemporal pattern of intracellular Ca 2+ signaling. In WT cells, transient ER Ca 2+ depletion triggered by localized IP3R-mediated Ca 2+ release is rapidly replenished through SOCE, driven by the coordinated activity of both STIM1 and STIM2. This balance of spatiotemporally separated localized Ca 2+ signals from both IP3Rs and SOCE supports efficient cell migration. In cells lacking either STIM1 or STIM2, the remaining isoform functions near its activation threshold due to lower ER Ca 2+ stores. As a result, even slight ER Ca 2+ depletion, such as that initiated by IP3R activity, activates the residual STIM protein, shifting IP3R-mediated Ca 2+ signals from localized to diffuse/global Ca 2+ release mode. This altered Ca 2+ pattern disrupts the finely tuned signaling required for cell migration, leading to impaired motility. In cells lacking both STIM isoforms, this feedback mechanism is lost entirely, allowing IP3Rs to continue generating localized Ca 2+ signals unmodulated by STIM proteins. Restoration of spatially confined Ca 2+ signaling supports the maintenance of cell migration despite the complete loss of STIM-mediated SOCE. Created with https://BioRender.com .

Article Snippet: Primary antibodies used were for IP3R-2 (1:500, sc-398434; Santa Cruz Biotechnology), IP3R-3 (1:2,000, BD 610312; BD Transduction Laboratories), or STIM1 (1:1,000, ACC-063; Alomone).

Techniques: Migration, Activity Assay, Activation Assay