Figures and data
Lysosomal Ca2+ and mitochondrial ROS in inflammasome. (A) Perilysosomal fluorescence after applying GPN to GCaMP3-ML1-transfected BMDMs treated with LP for a total of 4 h including LPS pretreatment for 3 h (left) (actual LP treatment time is 1 h). Peak fluorescence (right) (n=6). (B) [Ca2+]Lys in OGBD-loaded Mφs treated with LP for a total of 4 h including LPS pretreatment for 3 h (right). Representative fluorescence images (left) (n=8). (C) [Ca2+]i in Mφs treated with LP for a total of 4 h including LPS pretreatment for 3 h, determined using Fluo-3-AM staining (middle) or Fura-2 (right). Representative Fluo-3 images (left). (n=7 for BSA; n=6 for LPS; n=13 for LP). (D) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone or PA alone for 21 h, or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of BAPTA-AM (n=3) (left). Immunoblotting (IB) of lysate of Mφs treated with LPS alone or PA alone for 21 h, or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of BAPTA-AM or NAC, using indicated Abs (right). (E) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone or PA alone for 21 h, or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of MitoTEMPOL (n=3). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student’s t-test (A, B), one-way ANOVA with Tukey’s test (C), or two-way ANOVA with Sidak test (D, E) (ns, not significant). Scale bars, 20 μm. The online version of this article includes the following figure supplement for figure 1: Figure supplement 1. Mitochondrial ROS in inflammasome activation by LPS+PA (LP).
Lysosomal Ca2+ efflux through TRPM2 in inflammasome. (A) [Ca2+]i in Mφs treated with LPS alone for 4 h or with LP for a total of 4 h including LPS pretreatment for 3 h, determined using Fluo-3-AM (middle) or Fura-2 (right). Representative Fluo-3 images (left). (n=8 for Fluo-3-AM; n=9 for Fura-2). (B) [Ca2+]Lys in Mφs treated with LP for a total of 4 h including LPS pretreatment for 3 h, determined by OGBD loading (right). Representative fluorescence images (left). (n=5) (C) IL-1β ELISA of culture supernatant (upper) and IB of cell lysate using indicated Abs after treatment of Mφs with LPS alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 (lower). (n=4) (D) The number of ASC specks in Mφs treated with LP for a total of 21 h including LPS pretreatment for 3 h, determined by immunofluorescence using anti-ASC Ab (right). Representative confocal images (left). (n=4 for Trpm2+/+:BSA; n=5 for Trpm2+/+: LP; n=5 for Trpm2-/-:BSA; n=7 for Trpm2-/-:LP) Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A, B, D), or two-way ANOVA with Sidak test or Bonferroni test (C). Scale bars, 20 μm.
The online version of this article includes the following figure supplement for figure 2: Figure supplement 1. Effect of CD38 inhibitors and Trpm2 KO on inflammasome.
Ameliorated metabolic inflammation by Trpm2 KO. (A) Nonfasting blood glucose of mice on normal chow diet (NCD) (n=5 each) or HFD (n=8 each). (*: comparison between Trpm2+/+ and Trpm2-/- mice on HFD). (B) IPGTT after NCD (n=5 each) or HFD (n=8 each) feeding for 8 weeks (left). AUC (right). (C) The number of CLS in WAT after HFD feeding for 8 weeks (right). Representative H&E sections (left). (scale bar, 50 μm) (n=8 each). (D) The number of ASC specks in WAT after HFD feeding for 8 weeks, determined by immunofluorescence using anti-ASC Ab (right). Representative confocal images (left). (scale bar, 20 μm) (insets, magnified) (n=7 each). (E) IB of SVF of WAT after HFD feeding for 8 weeks using indicated Abs. (n=3). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by two-way ANOVA (B) or two-tailed Student’s t-test (A, C, D).
The online version of this article includes the following figure supplement for figure 3: Figure supplement 1. Plasma membrane TRPM2 current in Mφs and metabolic profile of Trpm2-KO mice.
ER→lysosome Ca2+ refilling in inflammasome. (A) [Ca2+]ER in GEM-CEPIA1er- (left) or D1ER-transfected BMDMs (right) treated with LP for 1 h without extracellular Ca 2+ after LPS pretreatment for 3 h. (n=26 for BSA; n=25 for LP). (B) BMDMs transfected with GEM-CEPIA1er and loaded with OGBD were treated with LP for 1 h after LPS pretreatment for 3 h (right) or BSA alone for 4 h (left). Tracing of [Ca2+]Lys and [Ca2+]ER after change to a fresh medium without extracellular Ca2+. (n=4 for BSA; n=4 for LP). (C) OGBD-loaded Mφs were treated with LP for 1 h after LPS pretreatment for 3 h. Recovery of [Ca2+]Lys after change to a fresh medium with or without Xestospongin C (Xesto C), dantrolene (Dan) or TPEN (right). Representative confocal images (left) (n=9 for BSA; n=8 for LP; n=9 for LP+Recovery; n=6 for LP+Recovery+Xesto C; n=5 for LP+Recovery+Dan; n=8 for LP+Recovery+TPEN). (D) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone or PA alone for 21 h, or with LP for a total of 21 h including LPS pretreatment for 3 h, in the presence or absence of Xesto C (n=4). (E) PLA in Mφs treated with LPS alone or PA alone for 21 h, or LP for a total of 21 h including LPS pretreatment for 3 h, using Abs to VAPA and ORP1L (right). Representative fluorescence images (left) (n=4). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by two-tailed Student’s t-test (A), one-way ANOVA with Tukey’s test (C, E) or two- way ANOVA with Sidak test (D). Scale bar, 20 μm.
The online version of this article includes the following figure supplement for figure 4: Figure supplement 1. Store-operated Ca2+ entry (SOCE) in inflammasome by LP.
Coupling of K+ efflux and Ca2+ influx in inflammasome. (A) [Ca2+]ER in D1ER- transfected BMDMs treated with LP for a total of 4 h including LPS pretreatment for 3 h at [K+]e of 5.4 or 60 mM (n=21 each). (B) OGBD-loaded Mφs were treated with LP for a total of 4 h including LPS pretreatment for 3 h. Recovery of [Ca2+]Lys in a fresh medium with 5.4 or 60 mM K+ (right). Representative fluorescence images (left) (n=7 for BSA; n=6 for LP; n=7 for LP+Recovery:5.4 mM K+; n=6 for LP+Recovery:60 mM K+). (C) [Ca2+]i in Mφs treated with LP for a total of 4 h including after LPS pretreatment for 3 h in a medium with 5.4 or 60 mM K+ (right). Representative Fluo-3 images (left) (n=14 for BSA; n=8 for LP:5.4 mM K+; n=6 for LP:60 mM K+). (D) [Ca2+]i in Mφs treated with LP for 1 h in the presence or absence of BTP2 or TRAM-34 after LPS pretreatment for 3 h, determined using Fluo-3-AM (middle) or Fura-2 (right). Representative Fluo-3 images (left) (For Fluo-3-AM, n=8) (For Fura-2, n=13). (E) [K+]i after LP treatment for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of BTP2 or TRAM-34 (right). Representative Potassium Green-2 images (left) (n=5). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, ** p< 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A-E). Scale bar, 20 μm.
The online version of this article includes the following figure supplement for figure 5: Figure supplement 1. Effect of high extracellular K+ and inhibitors of K+ efflux channels on inflammasome.
Role of KCa3.1 Ca2+-activated K+ channel in inflammasome. (A-D) Nystatin- perforated patch clamp and slope conductance of TRAM-34-sensitive I/V curve in Kcnn4+/+ (A, B) or Kcnn4-/- Mφs (C, D) treated with LPS alone for 4 h or with LP for a total of 4 h including LPS pretreatment for 3 h (B, D). Representative I/V curves (A, C). (n=13 for each Kcnn4+/+ group; n=4 for Kcnn4-/- :BSA; n=5 for Kcnn4-/- :LPS; n=4 for Kcnn4-/- :LP). (E) IL- 1β ELISA of culture supernatant (upper) and IB of cell lysate using indicated Abs (lower) after treating Kcnn4+/+ or Kcnn4-/- Mφs with PA alone or LPS alone for 21 h, or LP for a total of 21 h including LPS pretreatment for 3 h. (n=3). (F) [K+]i in Kcnn4+/+ or Kcnn4-/- Mφs treated with LP for a total of 21 h including LPS pretreatment for 3 h (right). Representative Potassium Green-2 images (left). (n=5). (G) OGBD-loaded BMDMs were treated with LP for a total of 4 h including LPS pretreatment for 3 h. [Ca2+]Lys recovery after LP removal with or without TRAM-34 (right). Representative fluorescence images (left). (n=11 for BSA; n=8 for LP; n=6 for LP:(-); n=9 for LP:TRAM-34). (H) BN gel electrophoresis and subsequent IB of lysate of Mφs treated with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h using indicated Ab. (I) IB of SVF of WAT from mice fed HFD for 8 weeks using indicated Abs. (n=3). (J) The number of ASC specks in WAT of mice of (I) identified by ASC immunofluorescence (right). Representative ASC specks (red arrow heads) (left). (scale bar, 20 μm) (n=28 for Kcnn4+/+ :HFD; n=22 for Kcnn4-/- :HFD). (K) The number of CLS in WAT of mice of (I) identified by F4/80 immunohistochemistry (lower). Representative F4/80 immunohistochemistry (upper). (scale bar, 50 μm) (n=12 for Kcnn4+/+ :HFD; n=11 for Kcnn4-/- :HFD). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (B, D, F, G), or two-way ANOVA with Sidak test (E). Scale bar, 20 μm.
The online version of this article includes the following figure supplement for figure 6: Figure supplement 1. Effects of Kcnn4 KO on metabolic profile and inflammasome.
Mechanism of Ca2+-mediated inflammasome. (A) IB of BMDMs treated with LPS alone without PA (‘- PA’) for indicated time period (left half) or with ‘PA’ (together with LPS) for indicated time period after LPS pretreatment for 3 h (right half) (hence, the numbers indicating LPS treatment time in the right half is 3 + PA treatment time), using indicated Abs. (B) IB of BMDMs treated with LPS alone for 0.5 h or ‘PA’ (together with LPS) for 1 or 18 h after LPS pretreatment for 3 h (hence, the numbers indicating LPS treatment time of 4 or 21 h is 3 + PA treatment time), using indicated Abs. (C) BMDMs were treated with LPS alone for 0.5 h, ‘PA’ (together with LPS) for 1 or 18 h after LPS pretreatment for 3 h (hence, the numbers indicating LPS treatment time of 4 or 21 h is 3 + PA treatment time) or nigericin for 45 min after LPS pretreatment for 3 h. IB using indicated Abs after DSS crosslinking. (D) BMDMs were treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of ASK1 (selonsertib) or JNK inhibitor (SP600125). IB using indicated Abs after DSS crosslinking. (E) IL-1β ELISA of culture supernatant after treating BMDMs with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of selonsertib or SP600125. (F-H) IB using indicated Abs (F, G) and IL-1β ELISA of culture supernatant (H) after treating BMDMs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of KN-93 or -92. (n=3). Data shown as means ± SEM from more than 3 independent experiments. ***p< 0.001 by two- way ANOVA with Tukey’s test (E, H).
The online version of this article includes the following figure supplement for figure 7: Figure supplement 1. Effect of Kcnn4 KO and inhibitor of TAK1 or ASK1 on activation of inflammasome and JNK.
Graphic summary. LP, an effector combination activating inflammasome related to metabolic inflammation, induces generation of mitochondrial ROS, which activates TRPM2 channel on lysosome and releases lysosomal Ca2+. ER→lysosome Ca2+ refilling facilitated by ER-lysosome tethering replenishes diminished lysosomal Ca2+ content and supports sustained lysosomal Ca2+ release. ER emptying due to ER→lysosome Ca2+ refilling activates SOCE. SOCE, in turn, is positively modulated by K+ efflux through KCa3.1, a Ca2+-activated K+ efflux channel, mediating hyperpolarization-induced acceleration of extracellular Ca2+ influx. Ca2+ release from lysosome activates CaMKII, which induces delayed activation of ASK1 and JNK. Delayed JNK activation leads to ASC phosphorylation and oligomerization, leading to formation of inflammasome complex together with NLRP3 and NEK7. K+ efflux changes intracellular milieu and induces structural changes of NLRP3 or NLRP3 binding to PI(4)P on dispersed Golgi network, facilitating inflammasome activation. Golgi complex and microtubule-organizing center (MTOC) are not shown for clarity. (CASP1, caspase 1)
Mitochondrial ROS in inflammasome activation by LPS+PA (LP). (A) Cellular ROS in peritoneal Mφs treated with LPS alone or PA alone for 21 h, or LP for a total of 21 h including LPS pretreatment for 3 h, determined by confocal microscopy after CM-H2DCFDA loading (right). Representative confocal images (left) (scale bar, 50 μm). (B) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone or PA alone for 21 h, or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of NAC (n=3). (C) [Ca2+]i in Mφs treated with LPS alone for 4 h or with LP for a total of 4 h including LPS pretreatment for 3 h in the presence or absence of NAC, determined by confocal microscopy after Fluo-3-AM loading (middle) or ratiometric measurement after Fura-2 loading (right). Representative Fluo-3 fluorescence images (left) (scale bar, 20 μm) (n=13 for BSA, Fluo-3-AM; n=12 for LPS, Fluo-3-AM; n=26 for LP, Fluo-3-AM; n=17 for LP+NAC, Fluo-3-AM; n=19 for BSA, Fura-2; n=23 for LP, Fura-2; n=23 for LP+BAPTA- AM, Fura-2; n=21 for LP+NAC, Fura-2). (D) Mitochondrial ROS in Mφs treated with PA alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of MitoTEMPOL, determined by flow cytometry after MitoSOX staining (right). Representative histograms (left) (n=3). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A, C, D), or two-way ANOVA with Sidak test (B).
Effect of CD38 inhibitors and Trpm2 KO on inflammasome. (A) Confocal microscopy was conducted after immunofluorescence staining of BMDMs using anti-TRPM2 and LAMP2 Abs. Yellow spots indicate TRPM2 on lysosome. (Rectangles were magnified) (B) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of apigenin or quercetin (n=3). (C) [Ca2+]i in Mφs treated with LPS alone for 4 h or with LP for a total of a 4 h including LPS pretreatment for 3 h in the presence or absence of apigenin or quercetin, determined by confocal microscopy after Fluo-3-AM loading (lower left) or ratiometric measurement after Fura-2 loading (lower right). Representative Fluo-3 fluorescence images (upper) (n=4 for BSA, Fluo-3-AM; n=4 for LP, Fluo-3-AM; n=4 for LP+apigenin, Fluo-3-AM; n=4 for LP+quercetin, Fluo-3-AM; n=13 for BSA, Fura-2; n=16 for LP, Fura-2; n=20 for LP+apigenin, Fura-2; n=13 for LP+quercetin, Fura-2). (D) Cellular ROS in Mφs from Trpm2+/+ or Trpm2-/- mice treated with LP for a total of 21 h including LPS pretreatment for 3 h, determined by CM-H2DCFDA staining (right). Representative fluorescence images (left) (n=4). (E) IL-1β ELISA of culture supernatant of Mφs from Trpm2+/+ or Trpm2-/- mice treated with PA, nigericin, ATP, L-leucyl-L-leucine methyl ester (LLOMe) and MSU for 18 h, 45 min, 1 h, 45 min and 3 h, respectively, after LPS pretreatment for 3 h (n=3). Data shown as means ± SEM from more than 3 independent experiments. ***p < 0.001 by one-way ANOVA with Tukey’s test (C,D) or two-way ANOVA with Tukey’s test (B,E). Scale bar, 20 μm.
Plasma membrane TRPM2 current in Mφs and metabolic profile of Trpm2-KO mice. (A) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone or PA alone for 21 h, or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of ACA (n=3). (B) Whole-cell patch clamp recording with 200 μM of cyclic ADP-ribose (cADPR) in pipette solution. At - 60 mV of holding voltage, development of inward current representing activation of TRPM2 by diffusion of cADPR to the cytosol was monitored in Mφs treated with carrier alone (BSA), PA alone or LPS alone for 4 h, or LP for a total of 4 h including LPS pretreatment for 3 h. Twenty μM of N[(p[amylcinnamoyl)anthranilic acid (ACA), a selective inhibitor of TRPM2, was applied to bath solution after confirming the steady-state activity inward current. (C) Amplitude of cADPR-induced inward current (b-c in B) (left group of the bar graph), and that of basal inward current inhibited by ACA (a-c in B) (right group of the bar graph) (n=19 for BSA; n=9 for PA; n=10 for LPS; n=9 for LP). (D) [Ca2+]i in Mφs from Trpm2+/+ and Trpm2-/- mice treated with LP for 1 h in the presence or absence of bafilomycin A1 emptying lysosomal Ca2+ reservoir after LPS pretreatment for 3 h, determined by ratiometric measurement after Fura-2 loading (n=10 for Trpm2+/+:BSA; n=27 for Trpm2+/+:LP; n=17 for Trpm2+/+:LP+BafA1; n=10 for Trpm2-/-:BSA; n=11 for Trpm2-/-:LP; n=9 for Trpm2-/-:LP+BafA1) (BafA1, bafilomycin A1). (E) Body weight of Trpm2+/+ and Trpm2-/- mice on NCD (n=5) or HFD (n=8). (F) HOMA-IR index in Trpm2+/+ and Trpm2-/- mice fed HFD for 8 weeks (n=5 for Trpm2+/+; n=8 for Trpm2-/-). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (C, D, F) or two-way ANOVA with Tukey’s test (E).
Store-operated Ca2+ entry (SOCE) in inflammasome by LP. (A-C) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone or PA alone for 21 h, or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of EGTA (A) (n=4), 2-APB (B) (n=4) or BTP2 (C) (n=3). (D) [Ca2+]ER in D1ER-transfected BMDMs treated with LP for 1 h in the presence or absence of BTP2 without removal of extracellular Ca2+ after LPS pretreatment for 3 h (n=19 for BSA; n=16 for LP; n=24 for LP+BTP2). (E) STIM1 aggregation (white arrows) and colocalization with ORAI1 (yellow arrows) at the middle and bottom levels of BMDMs that were transfected with YFP- STIM1 together with mCherry-Orai1 and then treated with LP in Ca2+-free KRB buffer for 1 h after LPS pretreatment for 3 h, determined by confocal microscopy (scale bar, 5 mm). Data shown as means ± SEM from more than 3 independent experiments. **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (D) or two-way ANOVA with Sidak test (A, B, C). Scale bar, 5 μm.
Effect of high extracellular K+ and inhibitors of K+ efflux channels on inflammasome. (A) [K+]i in Mφs treated with LPS alone or PA alone for 21 h, or LP for a total of 21 h including LPS pretreatment for 3 h (right), determined by confocal microscopy after Potassium Green-2-AM loading. Representative Potassium Green-2 fluorescence images (left) (n=4 for BSA; n=2 for PA; n=4 for LPS; n=4 for LP). (B) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h, at [K+]e of 5.4 (K+ concentration in RPMI medium) or 60 mM (n=3 each). (C, D) IL-1β ELISA of culture supernatant after treating Mφs with LPS alone or PA alone for 21 h, or LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of charybdotoxin (CTX) or TRAM-34 (C) (n=3), or several K+ efflux channel inhibitors (D) (n=3). (E) [Ca2+]ER in D1ER-transfected BMDMs after LP treatment for 1 h in the presence or absence of TRAM-34 without extracellular Ca2+ removal after LPS pretreatment for 3 h (n=16). Data shown as means ± SEM from more than 3 independent experiments. **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A, E) or two-way ANOVA with Sidak test or with Tukey’s test (B, C, D). Scale bar, 20 μm.
Effects of Kcnn4 KO on metabolic profile and inflammasome. (A) Real-time RT-PCR of Kcnn4 using mRNA from Mφs treated with PA alone or LPS alone for 21 h, or LP for a total of 21 h including LPS pretreatment for 3 h (n=3). (B) Real-time RT-PCR of Kcnn4 using mRNA from Mφs of Kcnn4+/+ or Kcnn4-/- mice (n=3). (C) TNF-α and IL-6 ELISA of culture supernatant after treating Mφs from Kcnn4+/+ or Kcnn4-/- mice with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h (n=3). (D) PLA in Mφs treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of BAPTA-AM, using Abs specific for KCNN4 and ORAI1 (right). Representative fluorescence images (left) (scale bar, 20 μm) (n=10 for BSA; n=16 for LP; n=10 for BSA+BAPTA-AM; n=16 for LP+BAPTA-AM). (E) IL-1β ELISA of culture supernatant after treating Mφs from Kcnn4+/+ or Kcnn4-/- mice with PA, nigericin, ATP, LLOMe and MSU for 18 h, 45 min, 1 h, 45 min and 3 h, respectively, after LPS pretreatment for 3 h (n=6). (F) Immunoprecipitation (IP) of Mφs treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of TRAM-34 using anti-NEK7 Ab and protein G beads. After bead heating in a sample buffer, collected supernatant was subjected to immunoblotting (IB) using indicated Abs. (red arrow, band of the correct size) (G) Immunoprecipitation of Mφs from Kcnn4+/+ or Kcnn4-/- mice treated with LP for a total of 21 h including LPS pretreatment for 3 h using anti-NEK7 Ab and protein G beads. After bead heating in a sample buffer, collected supernatant was subjected to IB using indicated Abs. (red arrow, band of the correct size) (H) Intraperitoneal glucose tolerance test (IPGTT) in Kcnn4+/+ and Kcnn4-/- mice fed HFD for 8 weeks (left). AUC (right) (n=9). Data shown as means ± SEM from more than 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s test (A, D), two-tailed Student’s t-test (B, H), or two-way ANOVA with Sidak test (C, E).
Effect of Kcnn4 KO and inhibitor of TAK1 or ASK1 on activation of inflammasome and JNK. (A) [Ca2+]i (left), [K+]i (middle) and [Ca2+]ER (right) determined by Flou-3-AM staining, Potassium Green-2-AM staining and D1ER transfection, respectively, after treatment with LP for a total of 4 or 21 h including LPS pretreatment for 3 h (n=10 for Fluo-3-AM; n=10 for Potassium Green-2-AM; n=50 for D1ER). (B) IB of lysate of Mφs from Kcnn4+/+ and Kcnn4-/- mice (left) or Trpm2+/+ and Trpm2-/- mice (right) treated with PA alone for 21 h, LPS alone for 21 h or LP for a total of 21 h including LPS pretreatment for 3 h, using indicated Abs. (C) IB of lysate of BMDMs treated with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of ASK1 (selonsertib) or JNK inhibitor (SP600125) using indicated Ab. (D) IL-1β ELISA of culture supernatant after treating BMDMs with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of 5Z-7-oxozeaenol (n=3). (E) IB of lysate of BMDMs treated with LPS alone for 21 h or with LP for a total of 21 h including LPS pretreatment for 3 h in the presence or absence of selonsertib using indicated Abs. Data shown as means ± SEM from more than 3 independent experiments*p < 0.05, **p < 0.01 and ***p < 0.001 by one- way ANOVA with Tukey’s test (A), or two-way ANOVA with Tukey’s test (D).
