Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin

doi: 10.3390/ijms241411692

Figure Lengend Snippet: TUDCA treatment inhibited BCG-infected THP-1 macrophage pyroptosis. THP-1 macrophages were pretreated for 2 h with TUDCA (5 mM) prior to BCG infection (MOI = 10) for 24 h. ( A ) The expression of Cleaved Caspase1, GSDMD-N, IL-1β, Cleaved-IL-1β and IL-18 was assessed by Western blotting. ( B ) TEM observation of cell morphology. Scale bar: 1 μm. Nucleus (N); mitochondria (Mi); green arrows: cell membrane intact; red arrow: cell membrane rupture; yellow arrow: ribosome loss; orange arrow: mitochondrial swelling; blue arrow: rough endoplasmic reticulum expansion; blue circle: chromatin dissolution. ( C , D ) Cell culture supernatant IL-1β and IL-18 concentrations were detected by ELISA. ( E ) Cell viability in THP-1 macrophages was detected using CCK-8 kit. ( F ) LDH activity in THP-1 macrophages was detected using LDH kit. Data are means ± SEM from triplicate experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Key reagents used included Fetal Bovine Serum (FBS, 10099141C, Gibco, Carlsbad, USA); RPMI-1640 (C11875500BT, Gibco, Carlsbad, CA, USA); β-mercaptoethanol (M8211, Solarbio, Beijing, China); PMA (P1585, Sigma-Aldrich, St. Louis, MO, USA); Middlebrook 7H9 Broth (M1315, BD, San Jose, CA, USA); Middlebrook ADC Enrichment (211887, BD, San Jose, CA, USA); M-PER (78501, Thermo Fisher Scientific, Waltham, MA, USA); Protease inhibitor Cocktail (78442, Sigma-Aldrich, St. Louis, MO, USA); BCA Protein Content Detection Kit (KGP902, KGI, Nanjing, China); anti-β-actin (20536-1-AP, Proteintech, Wuhan, China); anti-PREK (24390-1-AP, Proteintech, Wuhan, China); anti-GRP78 (11587-1-AP, Proteintech, Wuhan, China), HRP-labeled goat anti-rabbit IgG (SA00001-2, Proteintech, Wuhan, China); Fluorescein conjugated goat anti-rabbit IgG (SA00003-2, Proteintech, Wuhan, China); anti-IRE1α (3294S, Cell Signaling Technology, Danvers, MA, USA); anti-GSDMD-N (39754S, Cell Signaling Technology, Danvers, MA, USA); anti-Pro-Caspase1 (3866S, Cell Signaling Technology, Danvers, MA, USA); anti-ASC (13833S, Cell Signaling Technology, Danvers, MA, USA); anti-NLRP3 (15101S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-1β (12703S, Cell Signaling Technology, Danvers, MA, USA); anti-Cleaved-IL-1β (83186S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-18 (54943S, Cell Signaling Technology, Danvers, MA, USA); anti-ATF6 (DF6009, Affinity, Changzhou, China); anti-Cleaved Caspase1 (AF4005, Affinity, Changzhou, China); Normal Donkey Serum (SL050, Solarbio, Beijing, China); Hoechst 33,342 (C1028, Beyotime, Shanghai, China); Human IL-1 beta PicoKine ELISA Kit (EK0392, Boster, Wuhan, China); Human IL-18 PicoKine ELISA Kit (EK0864, Boster, Wuhan, China); CCK-8 Kit (abs50003, Absin, Shanghai, China); LDH Assay Kit (ab102526, Abcam, Cambridge, UK); TUDCA sodium (HY-19696A, MedChemExpress, Monmouth Junction, NJ, USA); MCC950 (HY-12815, MedChemExpress, Monmouth Junction, NJ, USA); Z-VAD-FMK (HY-16658B, MedChemExpress, Monmouth Junction, NJ, USA); VX-765 (S2228, Selleckchem, Houston, TX, USA).

Techniques: Infection, Expressing, Western Blot, Membrane, Dissolution, Cell Culture, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin

doi: 10.3390/ijms241411692

Figure Lengend Snippet: NLRP3 inflammasome-mediated pyroptosis in BCG-infected THP-1 macrophages. THP-1 macrophages were pretreated with MCC950 (10 μM) for 2 h and infected with BCG (MOI = 10) for 24 h. ( A – F ) The expression of Cleaved Caspase1, GSDMD-N, IL-1β, Cleaved-IL-1β and IL-18 protein levels were assessed by Western blotting ( G ) TEM observation of cell morphology. Scale bar: 1 μm. nucleus(N); mitochondria (Mi); green arrows: cell membrane intact; red arrow: cell membrane rupture; yellow arrow: ribosome loss; orange arrow: mitochondrial swelling; blue arrow: rough endoplasmic reticulum expansion; green circle: extravasation of cytoplasmic contents. ( H , I ) Cell culture supernatant IL-1β and IL-18 concentrations were detected by ELISA. ( J ) THP-1 macrophage viability was detected using CCK-8 kit. ( K ) LDH activity in THP-1 macrophages was detected using LDH kit. Data are means (±SEM) of three independent experiments. * p < 0.05; *** p < 0.001.

Article Snippet: Key reagents used included Fetal Bovine Serum (FBS, 10099141C, Gibco, Carlsbad, USA); RPMI-1640 (C11875500BT, Gibco, Carlsbad, CA, USA); β-mercaptoethanol (M8211, Solarbio, Beijing, China); PMA (P1585, Sigma-Aldrich, St. Louis, MO, USA); Middlebrook 7H9 Broth (M1315, BD, San Jose, CA, USA); Middlebrook ADC Enrichment (211887, BD, San Jose, CA, USA); M-PER (78501, Thermo Fisher Scientific, Waltham, MA, USA); Protease inhibitor Cocktail (78442, Sigma-Aldrich, St. Louis, MO, USA); BCA Protein Content Detection Kit (KGP902, KGI, Nanjing, China); anti-β-actin (20536-1-AP, Proteintech, Wuhan, China); anti-PREK (24390-1-AP, Proteintech, Wuhan, China); anti-GRP78 (11587-1-AP, Proteintech, Wuhan, China), HRP-labeled goat anti-rabbit IgG (SA00001-2, Proteintech, Wuhan, China); Fluorescein conjugated goat anti-rabbit IgG (SA00003-2, Proteintech, Wuhan, China); anti-IRE1α (3294S, Cell Signaling Technology, Danvers, MA, USA); anti-GSDMD-N (39754S, Cell Signaling Technology, Danvers, MA, USA); anti-Pro-Caspase1 (3866S, Cell Signaling Technology, Danvers, MA, USA); anti-ASC (13833S, Cell Signaling Technology, Danvers, MA, USA); anti-NLRP3 (15101S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-1β (12703S, Cell Signaling Technology, Danvers, MA, USA); anti-Cleaved-IL-1β (83186S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-18 (54943S, Cell Signaling Technology, Danvers, MA, USA); anti-ATF6 (DF6009, Affinity, Changzhou, China); anti-Cleaved Caspase1 (AF4005, Affinity, Changzhou, China); Normal Donkey Serum (SL050, Solarbio, Beijing, China); Hoechst 33,342 (C1028, Beyotime, Shanghai, China); Human IL-1 beta PicoKine ELISA Kit (EK0392, Boster, Wuhan, China); Human IL-18 PicoKine ELISA Kit (EK0864, Boster, Wuhan, China); CCK-8 Kit (abs50003, Absin, Shanghai, China); LDH Assay Kit (ab102526, Abcam, Cambridge, UK); TUDCA sodium (HY-19696A, MedChemExpress, Monmouth Junction, NJ, USA); MCC950 (HY-12815, MedChemExpress, Monmouth Junction, NJ, USA); Z-VAD-FMK (HY-16658B, MedChemExpress, Monmouth Junction, NJ, USA); VX-765 (S2228, Selleckchem, Houston, TX, USA).

Techniques: Infection, Expressing, Western Blot, Membrane, Cell Culture, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin

doi: 10.3390/ijms241411692

Figure Lengend Snippet: BCG infection promotes THP-1 macrophages pyroptosis via both canonical and noncanonical pathways. Cells were pretreated with 50 μM of VX-765 or 50 μM of Z-VAD-FMK for 2 h; they were then infected with BCG (MOI = 10) for 24 h. ( A – E ) The expression of GSDMD-N, IL-1β, Cleaved-IL-1β and IL-18 was measured by Western blotting. ( F , G ) Cell culture supernatant IL-1β and IL-18 concentrations were detected by ELISA. ( H ) LDH activity in THP-1 macrophages was detected using LDH kit. The data are presented as means ± SEM from triplicate experiments. * p < 0.05; *** p < 0.001.

Article Snippet: Key reagents used included Fetal Bovine Serum (FBS, 10099141C, Gibco, Carlsbad, USA); RPMI-1640 (C11875500BT, Gibco, Carlsbad, CA, USA); β-mercaptoethanol (M8211, Solarbio, Beijing, China); PMA (P1585, Sigma-Aldrich, St. Louis, MO, USA); Middlebrook 7H9 Broth (M1315, BD, San Jose, CA, USA); Middlebrook ADC Enrichment (211887, BD, San Jose, CA, USA); M-PER (78501, Thermo Fisher Scientific, Waltham, MA, USA); Protease inhibitor Cocktail (78442, Sigma-Aldrich, St. Louis, MO, USA); BCA Protein Content Detection Kit (KGP902, KGI, Nanjing, China); anti-β-actin (20536-1-AP, Proteintech, Wuhan, China); anti-PREK (24390-1-AP, Proteintech, Wuhan, China); anti-GRP78 (11587-1-AP, Proteintech, Wuhan, China), HRP-labeled goat anti-rabbit IgG (SA00001-2, Proteintech, Wuhan, China); Fluorescein conjugated goat anti-rabbit IgG (SA00003-2, Proteintech, Wuhan, China); anti-IRE1α (3294S, Cell Signaling Technology, Danvers, MA, USA); anti-GSDMD-N (39754S, Cell Signaling Technology, Danvers, MA, USA); anti-Pro-Caspase1 (3866S, Cell Signaling Technology, Danvers, MA, USA); anti-ASC (13833S, Cell Signaling Technology, Danvers, MA, USA); anti-NLRP3 (15101S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-1β (12703S, Cell Signaling Technology, Danvers, MA, USA); anti-Cleaved-IL-1β (83186S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-18 (54943S, Cell Signaling Technology, Danvers, MA, USA); anti-ATF6 (DF6009, Affinity, Changzhou, China); anti-Cleaved Caspase1 (AF4005, Affinity, Changzhou, China); Normal Donkey Serum (SL050, Solarbio, Beijing, China); Hoechst 33,342 (C1028, Beyotime, Shanghai, China); Human IL-1 beta PicoKine ELISA Kit (EK0392, Boster, Wuhan, China); Human IL-18 PicoKine ELISA Kit (EK0864, Boster, Wuhan, China); CCK-8 Kit (abs50003, Absin, Shanghai, China); LDH Assay Kit (ab102526, Abcam, Cambridge, UK); TUDCA sodium (HY-19696A, MedChemExpress, Monmouth Junction, NJ, USA); MCC950 (HY-12815, MedChemExpress, Monmouth Junction, NJ, USA); Z-VAD-FMK (HY-16658B, MedChemExpress, Monmouth Junction, NJ, USA); VX-765 (S2228, Selleckchem, Houston, TX, USA).

Techniques: Infection, Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay