Table S2 . Also, see , , and show representative images used for calculation. (H) MTT conversion to formazan at day 2 of PMA-differentiated THP-1 cells. Cells were treated with PMA for 24 h followed by being cultured in PMA-free, containing indicated stimulants media for 24 h. Unstimulated cells are used as normalization, set as 100%. Data shown are mean ± SEM of 3 independent experiments and each dot represents an independent experiment. DMSO is used as a control for CZ-48 and it’s also treated in cells shown as LPS. Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗∗∗∗ p < 0.0001. See also
and , , and . " width="100%" height="100%">
Journal: iScience
Article Title: SARM1 regulates pro-inflammatory cytokine expression in human monocytes by NADase-dependent and -independent mechanisms
doi: 10.1016/j.isci.2024.109940
Figure Lengend Snippet: Characterisation of expression of enzymatically active and inactive SARM1 in monocytes (A) Schematic of SARM1 domains and NADase enzymatic activity. The TIR domain has NADase activity and E642 is essential for this enzymatic activity. SARM1 forms an octamer, which is dependent on the SAM. To activate SARM1’s NADase activity, dimerisation of TIR domains within the octamer is required, facilitated by a conformation change which distances the inhibitory ARM domain from the TIR domain. Active SARM1 cleaves NAD + and produces NAM and cADPR. (B) Left panel, immunoblot showing stably expressing SARM1-FLAG protein in THP-1 cells stained with α-FLAG antibody. EV; empty vector control, SARM1-WT and SARM1-E642A. β-actin was used as a loading control. Representative of 4 experiments. Right panel, 4 independent experiments were performed and the FLAG-SARM1/β-actin ratio normalised to SARM1-WT was quantified by ImageJ. Data shown are mean ± SEM (bar chart) and each dot represents an independent experiment. Data were tested with an RM one-way ANOVA; Dunnett’s multiple comparisons test. (C) NAD + was assessed at day 5 of PMA-differentiated THP-1 cells. THP-1 cells seeded in 10 cm dishes were treated with PMA for 24 h followed by being cultured in PMA-free media. Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate (3 dishes were prepared) or quadruplicate (4 dishes were prepared). Dots show the results of each dish. Data were tested with a one-way ANOVA; Tukey’s multiple comparisons test, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (D) cADPR were measured at day 3 of PMA-differentiated THP-1 cells. THP-1 cells seeded in 10 cm dishes were treated with PMA for 24 h followed by being cultured in PMA-free media. Data shown are mean ± SEM of 3 independent experiments, each performed in duplicate (2 dishes were prepared) or triplicate (3 dishes were prepared). Dots show the results of each dish. (E) Undifferentiated THP-1 cells were cultured in PMA-free media and MTT assay performed at indicated time points. MTT conversion to formazan at day 1 is used as normalization, set as 100% viability. Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate. Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test. (F) THP-1 cells were treated with PMA for 24 h followed by being cultured in PMA-free media and MTT assay performed at indicated time points. MTT conversion to formazan at day 1 is used as normalization, set as 100% viability. Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate. Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗ p < 0.05, ∗∗∗∗ p < 0.0001. Significances are shown as comparison with SARM1-WT stably expressing cells. (G) PI uptake of PMA-differentiated THP-1 cells was measured every 6 h from day 1 to day 7. Cells were treated with PMA at day 0 followed by replacing media with PMA-free, PI containing media at day 1, prior to starting the scanning of images. Data shown are mean ± SEM of 3 independent experiments, each performed in quadruplicate. (see section for the detail). Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, results are shown in Table S2 . Also, see , , and show representative images used for calculation. (H) MTT conversion to formazan at day 2 of PMA-differentiated THP-1 cells. Cells were treated with PMA for 24 h followed by being cultured in PMA-free, containing indicated stimulants media for 24 h. Unstimulated cells are used as normalization, set as 100%. Data shown are mean ± SEM of 3 independent experiments and each dot represents an independent experiment. DMSO is used as a control for CZ-48 and it’s also treated in cells shown as LPS. Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗∗∗∗ p < 0.0001. See also Table S2 and , , and .
Article Snippet: Primary antibodies: SARM1 (ProSci #3295, for immunoblots for primary monocytes; CST #D2M5I for all other immunoblots), FLAG (Sigma-Aldrich #F1804), NLRP3 (Adipogen #AG-20B-0014), ASC (Santa Cruz #sc-22514R), Caspase 1 (AdipoGen #AG-20B-0048-C100), Caspase 1 p20 (CST #D57A2), IL-1β (R&D #AF-201-NA), GSDMD (Sigma-Aldrich #HPA044487), β-actin (Sigma-Aldrich #A5316).
Techniques: Expressing, Activity Assay, Western Blot, Stable Transfection, Staining, Plasmid Preparation, Control, Cell Culture, MTT Assay, Comparison
Cell Signaling Technology Inc is a verified supplier 
Cell Signaling Technology Inc manufactures this product 
Figure S2 . " width="250" height="auto" />
Table S2 . Also, see , , and show representative images used for calculation. (H) MTT conversion to formazan at day 2 of PMA-differentiated THP-1 cells. Cells were treated with PMA for 24 h followed by being cultured in PMA-free, containing indicated stimulants media for 24 h. Unstimulated cells are used as normalization, set as 100%. Data shown are mean ± SEM of 3 independent experiments and each dot represents an independent experiment. DMSO is used as a control for CZ-48 and it’s also treated in cells shown as LPS. Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗∗∗∗ p < 0.0001. See also 
