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<t>SARM1-deficient</t> monocytes display enhanced TNF and IL-1β secretion after LPS stimulation (A) Relative expression of SARM1 mRNA was examined by qRT-PCR, normalised to primary monocytes. Data shown are mean ± SEM (bar chart) and each dot represents one donor (primary monocytes, 7 donors) or independent experiments (BLaER1 and THP-1, n = 5; 293T and SH-SY5Y: n = 6). (B) Immunoblot of SARM1 protein in various cell lines. Each image is cropped from the same membrane. Endogenous SARM1 expression was observed in BLaER1, 293T and SH-SY5Y cells but not in THP-1 cells. Also, BLaER1 SARM1 KO cells show no detectable SARM1 protein. Representative of 3 experiments. (C–J) BLaER1 control WT and SARM1 KO cells were differentiated into monocyte for 7 days, then stimulated with LPS (C–E and H) or CL075 (F, G, I, and J) for the indicated time (hours). Supernatants were assayed for TNF (C and F), CCL5 (D and G) and IL-1β (H and I) protein by ELISA. Relative TNF (E) and IL-1β (J) mRNA were assessed by qRT-PCR. Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate. (K and L) BLaER1 monocytes were stimulated with LPS for 4 h, following nigericin stimulation for 1 h. LDH release (K) was measured to assess pyroptosis and IL-1β secretion in the supernatant (L) was assessed by ELISA. Data shown are mean ± SEM of 3 independent experiments and each dot represents an independent experiment. Data were tested with a two-way ANOVA; Šídák’s multiple comparisons test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also <xref ref-type=

Figure S2 . " width="250" height="auto" />
Sarm1, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sarm1 - by Bioz Stars, 2026-06

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1) Product Images from "SARM1 regulates pro-inflammatory cytokine expression in human monocytes by NADase-dependent and -independent mechanisms"

Article Title: SARM1 regulates pro-inflammatory cytokine expression in human monocytes by NADase-dependent and -independent mechanisms

Journal: iScience

doi: 10.1016/j.isci.2024.109940

SARM1-deficient monocytes display enhanced TNF and IL-1β secretion after LPS stimulation (A) Relative expression of SARM1 mRNA was examined by qRT-PCR, normalised to primary monocytes. Data shown are mean ± SEM (bar chart) and each dot represents one donor (primary monocytes, 7 donors) or independent experiments (BLaER1 and THP-1, n = 5; 293T and SH-SY5Y: n = 6). (B) Immunoblot of SARM1 protein in various cell lines. Each image is cropped from the same membrane. Endogenous SARM1 expression was observed in BLaER1, 293T and SH-SY5Y cells but not in THP-1 cells. Also, BLaER1 SARM1 KO cells show no detectable SARM1 protein. Representative of 3 experiments. (C–J) BLaER1 control WT and SARM1 KO cells were differentiated into monocyte for 7 days, then stimulated with LPS (C–E and H) or CL075 (F, G, I, and J) for the indicated time (hours). Supernatants were assayed for TNF (C and F), CCL5 (D and G) and IL-1β (H and I) protein by ELISA. Relative TNF (E) and IL-1β (J) mRNA were assessed by qRT-PCR. Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate. (K and L) BLaER1 monocytes were stimulated with LPS for 4 h, following nigericin stimulation for 1 h. LDH release (K) was measured to assess pyroptosis and IL-1β secretion in the supernatant (L) was assessed by ELISA. Data shown are mean ± SEM of 3 independent experiments and each dot represents an independent experiment. Data were tested with a two-way ANOVA; Šídák’s multiple comparisons test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also <xref ref-type=

Figure S2 . " title="SARM1-deficient monocytes display enhanced TNF and IL-1β secretion after ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: SARM1-deficient monocytes display enhanced TNF and IL-1β secretion after LPS stimulation (A) Relative expression of SARM1 mRNA was examined by qRT-PCR, normalised to primary monocytes. Data shown are mean ± SEM (bar chart) and each dot represents one donor (primary monocytes, 7 donors) or independent experiments (BLaER1 and THP-1, n = 5; 293T and SH-SY5Y: n = 6). (B) Immunoblot of SARM1 protein in various cell lines. Each image is cropped from the same membrane. Endogenous SARM1 expression was observed in BLaER1, 293T and SH-SY5Y cells but not in THP-1 cells. Also, BLaER1 SARM1 KO cells show no detectable SARM1 protein. Representative of 3 experiments. (C–J) BLaER1 control WT and SARM1 KO cells were differentiated into monocyte for 7 days, then stimulated with LPS (C–E and H) or CL075 (F, G, I, and J) for the indicated time (hours). Supernatants were assayed for TNF (C and F), CCL5 (D and G) and IL-1β (H and I) protein by ELISA. Relative TNF (E) and IL-1β (J) mRNA were assessed by qRT-PCR. Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate. (K and L) BLaER1 monocytes were stimulated with LPS for 4 h, following nigericin stimulation for 1 h. LDH release (K) was measured to assess pyroptosis and IL-1β secretion in the supernatant (L) was assessed by ELISA. Data shown are mean ± SEM of 3 independent experiments and each dot represents an independent experiment. Data were tested with a two-way ANOVA; Šídák’s multiple comparisons test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S2 .

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Membrane, Control, Enzyme-linked Immunosorbent Assay

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 <xref ref-type=

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 . " title="Characterisation of expression of enzymatically active and inactive SARM1 in monocytes (A) Schematic of SARM1 domains and ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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 .

Techniques Used: Expressing, Activity Assay, Western Blot, Stable Transfection, Staining, Plasmid Preparation, Control, Cell Culture, MTT Assay, Comparison

SARM1 inhibition of TLR4-stimulated TNF mRNA induction is NADase-independent (A and B) Relative TNF (A) and IL-1β (B) mRNA were assessed by qRT-PCR, normalised to undifferentiated cells (left) or PMA-differentiated cells (right). Data shown are mean ± SEM of 6 independent experiments, each performed in triplicate or quadruplicate. Dots represent independent experiments (left). Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Figure Legend Snippet: SARM1 inhibition of TLR4-stimulated TNF mRNA induction is NADase-independent (A and B) Relative TNF (A) and IL-1β (B) mRNA were assessed by qRT-PCR, normalised to undifferentiated cells (left) or PMA-differentiated cells (right). Data shown are mean ± SEM of 6 independent experiments, each performed in triplicate or quadruplicate. Dots represent independent experiments (left). Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Techniques Used: Inhibition, Quantitative RT-PCR

SARM1 regulates IL-1β secretion through NADase-dependent and -independent mechanisms (A and B) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Supernatants were collected to measure IL-1β protein secretion by ELISA (Α) and LDH release (B). 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 < 0.0001. (C) Left panel, PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblot of supernatants from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Cleaved caspase-1 (p20), cleaved GSDMD (p30) and cleaved IL-1β (p17) are shown. Representative of 3 independent experiments. Right panel, 3 independent experiments were performed and Caspase 1 p20 was quantified by using ImageJ. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗ p < 0.05. (D) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblots of cell lysates from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Representative of 3 independent experiments. β-actin was used as a loading control. (E) Quantification of pro-IL-1β from (D). Pro-IL-1β/β-actin ratios were normalised to PMA-treated EV control cells without stimulation. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗ p < 0.05, ∗∗ p < 0.01. (F) Left panel, PMA differentiated THP-1 cells were stimulated with LPS and/or CZ-48 for 6 h. Immunoblots of pro-IL-1β and SARM1 (to show no effect by NADase in other proteins) from cell lysates of PMA-differentiated THP-1 stimulated with LPS and/or CZ-48. Representative of 3 independent experiments. β-actin was used as a loading control. Right panel, quantification of pro-IL-1β and significance is assessed as described in (E). ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Figure Legend Snippet: SARM1 regulates IL-1β secretion through NADase-dependent and -independent mechanisms (A and B) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Supernatants were collected to measure IL-1β protein secretion by ELISA (Α) and LDH release (B). 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 < 0.0001. (C) Left panel, PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblot of supernatants from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Cleaved caspase-1 (p20), cleaved GSDMD (p30) and cleaved IL-1β (p17) are shown. Representative of 3 independent experiments. Right panel, 3 independent experiments were performed and Caspase 1 p20 was quantified by using ImageJ. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗ p < 0.05. (D) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblots of cell lysates from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Representative of 3 independent experiments. β-actin was used as a loading control. (E) Quantification of pro-IL-1β from (D). Pro-IL-1β/β-actin ratios were normalised to PMA-treated EV control cells without stimulation. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗ p < 0.05, ∗∗ p < 0.01. (F) Left panel, PMA differentiated THP-1 cells were stimulated with LPS and/or CZ-48 for 6 h. Immunoblots of pro-IL-1β and SARM1 (to show no effect by NADase in other proteins) from cell lysates of PMA-differentiated THP-1 stimulated with LPS and/or CZ-48. Representative of 3 independent experiments. β-actin was used as a loading control. Right panel, quantification of pro-IL-1β and significance is assessed as described in (E). ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Control

Figure Legend Snippet:

Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Lactate Dehydrogenase Assay, Bicinchoninic Acid Protein Assay, Transfection, Control, Plasmid Preparation, Sequencing, Expressing, Software

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Figure S2 . " 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: SARM1-deficient monocytes display enhanced TNF and IL-1β secretion after LPS stimulation (A) Relative expression of SARM1 mRNA was examined by qRT-PCR, normalised to primary monocytes. Data shown are mean ± SEM (bar chart) and each dot represents one donor (primary monocytes, 7 donors) or independent experiments (BLaER1 and THP-1, n = 5; 293T and SH-SY5Y: n = 6). (B) Immunoblot of SARM1 protein in various cell lines. Each image is cropped from the same membrane. Endogenous SARM1 expression was observed in BLaER1, 293T and SH-SY5Y cells but not in THP-1 cells. Also, BLaER1 SARM1 KO cells show no detectable SARM1 protein. Representative of 3 experiments. (C–J) BLaER1 control WT and SARM1 KO cells were differentiated into monocyte for 7 days, then stimulated with LPS (C–E and H) or CL075 (F, G, I, and J) for the indicated time (hours). Supernatants were assayed for TNF (C and F), CCL5 (D and G) and IL-1β (H and I) protein by ELISA. Relative TNF (E) and IL-1β (J) mRNA were assessed by qRT-PCR. Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate. (K and L) BLaER1 monocytes were stimulated with LPS for 4 h, following nigericin stimulation for 1 h. LDH release (K) was measured to assess pyroptosis and IL-1β secretion in the supernatant (L) was assessed by ELISA. Data shown are mean ± SEM of 3 independent experiments and each dot represents an independent experiment. Data were tested with a two-way ANOVA; Šídák’s multiple comparisons test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S2 .

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, Quantitative RT-PCR, Western Blot, Membrane, Control, Enzyme-linked Immunosorbent Assay

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 .

Techniques: Expressing, Activity Assay, Western Blot, Stable Transfection, Staining, Plasmid Preparation, Control, Cell Culture, MTT Assay, Comparison

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: SARM1 inhibition of TLR4-stimulated TNF mRNA induction is NADase-independent (A and B) Relative TNF (A) and IL-1β (B) mRNA were assessed by qRT-PCR, normalised to undifferentiated cells (left) or PMA-differentiated cells (right). Data shown are mean ± SEM of 6 independent experiments, each performed in triplicate or quadruplicate. Dots represent independent experiments (left). Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Techniques: Inhibition, Quantitative RT-PCR

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: SARM1 regulates IL-1β secretion through NADase-dependent and -independent mechanisms (A and B) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Supernatants were collected to measure IL-1β protein secretion by ELISA (Α) and LDH release (B). 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 < 0.0001. (C) Left panel, PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblot of supernatants from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Cleaved caspase-1 (p20), cleaved GSDMD (p30) and cleaved IL-1β (p17) are shown. Representative of 3 independent experiments. Right panel, 3 independent experiments were performed and Caspase 1 p20 was quantified by using ImageJ. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗ p < 0.05. (D) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblots of cell lysates from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Representative of 3 independent experiments. β-actin was used as a loading control. (E) Quantification of pro-IL-1β from (D). Pro-IL-1β/β-actin ratios were normalised to PMA-treated EV control cells without stimulation. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗ p < 0.05, ∗∗ p < 0.01. (F) Left panel, PMA differentiated THP-1 cells were stimulated with LPS and/or CZ-48 for 6 h. Immunoblots of pro-IL-1β and SARM1 (to show no effect by NADase in other proteins) from cell lysates of PMA-differentiated THP-1 stimulated with LPS and/or CZ-48. Representative of 3 independent experiments. β-actin was used as a loading control. Right panel, quantification of pro-IL-1β and significance is assessed as described in (E). ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control

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:

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Lactate Dehydrogenase Assay, Bicinchoninic Acid Protein Assay, Transfection, Control, Plasmid Preparation, Sequencing, Expressing, Software

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