Journal: BMC Cardiovascular Disorders

Article Title: MicroRNA-218-5p regulates inflammation response via targeting TLR4 in atherosclerosis

doi: 10.1186/s12872-023-03124-y

Figure Lengend Snippet: Prediction and verification of miR-218-5p target genes. (A) . Complementary binding sites of miR-218-5p and TLR4. (B) . Luciferase reporter gene assay confirmed that miR-218-5p directly targets TLR4. (C) . TLR4 expression was increased in ox-LDL-induced THP-1 cells. *** P < 0.001 vs. Control group

Article Snippet: The human monocyte cell line THP-1 cells were purchased from ATCC.

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Expressing

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: miR-100-5p expression in BCG-infected THP-1 cells. ( A ) PMA-differentiated THP-1 cells were infected with BCG at MOIs of 2, 5, and 10 for 8 h. Total RNA was extracted at 24 hpi. The miR-100-5p expression level was measured by qRT-PCR normalized with U6 as the internal reference. ( B ) PMA-differentiated THP-1 cells were infected with BCG at an MOI of 10 for 8 h. Total RNA was extracted at 0, 12, and 24 hpi. The miR-100-5p expression level was measured by qRT-PCR normalized with U6 as the internal reference. Data are representative of three independent experiments with biological duplicates in each (( A , B ); mean ± s.e.m. for n = 3 duplicates). ** p < 0.01; *** p < 0.001 (ANOVA).

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Expressing, Infection, Quantitative RT-PCR

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: miR-100-5p inhibits LDH release after BCG infection. LDH assay in THP-1 cells transfected with NC mimic, miR-100-5p mimic ( A ) or NC inhibitor, miR-100-5p inhibitor ( B ) and infected with BCG (MOI = 10) for 8 h. Cell death was detected by medium LDH release assay at 0, 12, 24, and 48 hpi. Data are from three independent experiments with biological duplicates in each (( A , B ); mean ± s.e.m. for n = 3 duplicates). ** p < 0.01; *** p < 0.001.

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Infection, Lactate Dehydrogenase Assay, Transfection

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: Regulation of apoptosis of BCG-infected THP-1 cells by miR-100-5p. THP-1 cells were transfected with NC mimic, mimic, NC inhibitor, or miR-100-5p inhibitor and then infected with BCG (MOI = 10) for 8 h. ( A ) Cell apoptosis was detected by flow cytometry at 12 hpi. There were six groups: transfected NC mimic and infected BCG, transfected miR-100-5p mimic and infected BCG, transfected NC inhibitor and infected BCG, transfected miR-100-5p inhibitor and infected BCG, untransfected but infected BCG (BCG), and untransfected and uninfected (NC). ( B , C ) Cell total apoptosis rate with different groups. ( D ) Total proteins were collected at 12 hpi. Caspase-3 and Bcl-2 protein levels were detected by Western blotting assay. ( E , F ) Relative protein expression ratios were analyzed using ImageJ with normalization to β-actin. Data are from three independent experiments with biological duplicates in each (( A – C ); mean ± s.e.m. of n = 3 duplicates) or representative of three independent experiments ( D – F ). ** p < 0.01; *** p < 0.001 (Student’s t -test for one comparison or ANOVA for more than one comparison).

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Infection, Transfection, Flow Cytometry, Western Blot, Expressing

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: miR-100-5p increases PI3K and AKT phosphorylation. THP-1 cells were transfected with NC mimic, miR-100-5p mimic, NC inhibitor, or miR-100-5p inhibitor for 24 h and infected with BCG at an MOI of 10 for 8 h. ( A , B ) Total proteins were collected at 12 hpi. p-PI3K and p-AKT protein levels were detected by Western blotting assay. ( C , D ) Relative protein expression ratios were analyzed using ImageJ with normalization to GAPDH. Data are representative of three independent experiments (( C , D ), mean ± s.e.m.). * p < 0.05; *** p < 0.001.

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Transfection, Infection, Western Blot, Expressing

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: miR-100-5p promotes BCG survival in macrophages. THP-1 cells were transfected with NC mimic, miR-100-5p mimic, NC inhibitor, or miR-100-5p inhibitor for 24 h and infected with BCG at MOI 10 for 8 h. ( A , B ) Viable intracellular BCG in THP-1 cells transfected with miR-100-5p mimic or inhibitor compared to the negative control. ( C ) Percentage analysis of surviving intracellular bacteria based on 0 hpi with four groups. Data are from three independent experiments with biological duplicates in each (( A – C ); mean ± s.e.m. of n = 3 duplicates). ** p < 0.01; *** p < 0.001.

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Transfection, Infection, Negative Control

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: Screening and validation of miR-100-5p target genes. ( A ) miR-100-5p target gene prediction with five software and analyzed by Venn diagram. ( B ) Binding site of miR-100-5p with SMARCA5 WT or mut 3′-UTR as predicted by online prediction software. ( C ) Dual-luciferase reporter assay performed in HEK 293T cells. miR-100-5p mimic or NC mimic was cotransfected with dual-luciferase reporter plasmids, such as psiCHECK2 control, psiCHECK2-SMARCA5-wt, and psiCHECK-SMARCA5-mut. The ratio represents the Renilla to firefly (Rluc/Fluc) ratio in miR-100-5p mimic versus NC mimic. ( D ) Differential SMARCA5 expression between BCG-infected (MOI = 10) and uninfected cells measured by qRT-PCR at 0, 12, and 24 hpi. ( E , F ) THP-1 cells were transfected with NC mimic, miR-100-5p mimic, NC inhibitor, or miR-100-5p inhibitor for 24 h and infected with BCG at an MOI 10 for 8 h. SMARCA5 mRNA expression was detected by qRT-PCR at 12, 24, and 48 hpi. ( G – H ) SMARCA5 protein expression detected by Western blotting assay. The expression ratio was analyzed using ImageJ with normalization to β-actin. Data are from three independent experiments with biological duplicates in each (( C – F ); mean ± s.e.m. of n = 3 duplicates) or representative of three independent experiments (( G , H ), mean ± s.e.m). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Software, Binding Assay, Luciferase, Reporter Assay, Expressing, Infection, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: Construction of miR-100-5p and siSMARCA5 cotransfection model. ( A – C ) After transfection of three siRNAs, SMARCA5 mRNA and protein expression levels were detected by qRT-PCR and Western blotting. The protein expression ratios were analyzed using ImageJ with normalization to GAPDH. ( D ) Western blot detected SMARCA5 protein expression from THP-1 cells pretreated with miR-100-5p inhibitor, siSMARC5 and their negative controls infected with BCG. ( E ) Expression ratios analyzed using ImageJ with normalization to GAPDH. Data are from three independent experiments with biological duplicates in each (( A ); mean ± s.e.m. of n = 3 duplicates) or representative of three independent experiments (( B – E ), mean ± s.e.m). ** p < 0.01; *** p < 0.001.

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Cotransfection, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Infection

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: miR-100-5p inhibits apoptosis and promotes BCG survival via SMARCA5 in BCG-infected macrophages. THP-1 cells were cotransfected with NC inhibitor, miR-100-5p inhibitor, siSMARCA5, or control siRNA for 24 h and infected with BCG at an MOI 10 for 8 h. There were four groups: cotransfected NC inhibitor and siRNA control, cotransfected NC inhibitor and siSMARCA5, cotransfected miR-100-5p inhibitor and siRNA control, and cotransfected miR-100-5p inhibitor and siSMARCA5. ( A ) Medium LDH release detected at 12, 24, and 48 hpi with OD490 nm. ( B , C ) Cell apoptosis identified by flow cytometry at 12 hpi. The total apoptosis rate was analyzed by GraphPad Prism. ( D ) Caspase-3 and Bcl-2 protein levels detected by Western blotting assay. ( E , F ) Relative protein expression ratios analyzed using ImageJ with normalization to GAPDH. ( G , H ) Number of intracellular bacteria (CFU/mL) determined at 48 hpi. The percentage of surviving intracellular bacteria based on 0 hpi was analyzed. Data are from three independent experiments with biological duplicates in each (( A – C , G , H ); mean ± s.e.m. of n = 3 duplicates) or representative of three independent experiments (( D – F ), mean ± s.e.m). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Infection, Flow Cytometry, Western Blot, Expressing

Journal: Cells

Article Title: The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells

doi: 10.3390/cells12030476

Figure Lengend Snippet: Pattern of miR-100-5p-mediated apoptosis to clear BCG in macrophages. miR-100-5p was downregulated when THP-1 cells were infected with BCG, promoting SMARCA5 expression and inhibiting the PI3K/Akt signaling pathway. Thus, caspase-3 expression increased, resulting in cell apoptosis. BCG intracellular survival was reduced finally.

Article Snippet: Human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in 5% CO 2 .

Techniques: Infection, Expressing

Journal: Frontiers in Pharmacology

Article Title: Anti-inflammatory activities of Coleus forsteri (formerly Plectranthus forsteri ) extracts on human macrophages and chemical characterization

doi: 10.3389/fphar.2022.1081310

Figure Lengend Snippet: Inhibitory effect of C. forsteri extracts on cytokine production. Anti-inflammatory potential of C. forsteri cyclohexane (cPE) and ethanolic (ePE) plant extract at 25 μg/ml was evaluated on cytokines produced by PMA-THP-1 macrophages induced with LPS (1 μg/ml) for 24 h. Dexamethasone (DEX) at 127 nM was used as positive inhibitory control. Cytotoxicity of cPE and ePE was evaluated on LPS-induced PMA-THP-1 macrophages (A) as described in Materials and Methods. Cytokines IL-1β (B) , IL-6 (C) , IL-10 (D) and TNF-α (E) were quantified using ELISA technique. Mann-Whitney analyses were used for comparison between treatments compared to control or LPS condition. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001; ns, non-significant.

Article Snippet: The human THP-1 cell line (ATCC ® TIB-202™) and the monocyte-derived macrophage (MDM) primary culture were used as in vitro models to study the anti-inflammatory effect of C. forsteri extracts.

Techniques: Produced, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Danger signals released during cold ischemia storage activate NLRP3 inflammasome in myeloid cells and influence early allograft function in liver transplantation

doi: 10.1016/j.ebiom.2022.104419

Figure Lengend Snippet: eiOPS activates NLRP3 inflammasome in human myeloid cells. (a) Release of IL-1β by LPS-primed THP-1 cells after incubation with Celsior® or eiOPS for 4 or 16 h. Incubation for 30 min with potassium ionophore nigericin (10 μM) or ATP (3 mM) were used as positive control for NLRP3 activation. Presence of IL-1β in Celsior® and eiOPS before incubation with THP-1 cells was determined as negative control. n = 5. (b) Release of IL-1β by LPS-primed THP-1 cells after incubation with eiOPS for 16 h in the presence of caspase-1 inhibitor Ac-YVAD-AOM (100 μM) or NLRP3 inhibitor MCC950 (10 μM). n = 8. (c) Release of IL-1β by LPS-primed wild-type (WT), NLRP3 −/− , PYCARD −/− , and CASP1 −/− THP-1 cells after incubation with eiOPS for 16 h n = 6. (d) Representative microscopy images of ASC specks in THP-1 cells as detected by immunofluorescence, and percentage of THP-1 cells with at least one ASC speck. Quantification of intracellular ASC specks was done from 6 random ×20 images/conditions in at least 3 independent experiments. (e) Release of IL-1β by LPS-primed human primary monocytes after incubation with eiOPS for 16 h in the presence of Ac-YVAD-AOM (100 μM) or MCC950 (10 μM). Incubation with nigericin (10 μM) or ATP (3 mM) after LPS priming were used as canonical NLRP3 activation positive control. n = 5. Representative pictures in (d) are from 4 independent experiments. Results are presented as mean ± SEM; each sample was tested in duplicate; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001 [one-way ANOVA followed by Bonferroni test (b, c and e) or unpaired Student's t test (d)].

Article Snippet: The human macrophage THP-1 cell line was from the American Type Culture Collection (Cat# TIB-202, RRID: CVCL_0006).

Techniques: Incubation, Positive Control, Activation Assay, Negative Control, Microscopy, Immunofluorescence

Journal: eBioMedicine

Article Title: Danger signals released during cold ischemia storage activate NLRP3 inflammasome in myeloid cells and influence early allograft function in liver transplantation

doi: 10.1016/j.ebiom.2022.104419

Figure Lengend Snippet: Different DAMPs in eiOPS are able to activate NLRP3 inflammasome in human primary monocytes. (a) Correlation matrix between concentration of IL-1β released by LPS-primed THP-1 cells after incubation with eiOPS and concentrations of DAMPs present in OPS recovered after cold ischemia storage of liver n = 6. (b–e) Release of IL-1β by LPS-primed human primary monocytes after incubation with eiOPS for 16 h in the presence of specific P2X7R antagonist A438079 (50 μM) (b), phagocytosis inhibitor cytochalasin D (10 μM) or clathrin-mediated endocytosis inhibitor monodansylcadaverine (MDC, 250 μM) (c), specific scavenger of mitochondrial superoxide mitoTEMPO (500 μM) or anti-oxidant N -acetyl-L-cysteine (NAC, 10 mM) (d), and IL-18BP (100 ng/mL), anti-HMGB1, or mouse IgG isotype control (100 ng/mL) (e). Alternatively, eiOPS previously incubated with proteinase K (1 mg/mL) or uricase (2 U/mL) was used (b). n = 6. Results are presented as mean ± SEM; each sample was tested in duplicate; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001 [one-way ANOVA followed by Bonferroni test].

Article Snippet: The human macrophage THP-1 cell line was from the American Type Culture Collection (Cat# TIB-202, RRID: CVCL_0006).

Techniques: Concentration Assay, Incubation

Journal: eBioMedicine

Article Title: Danger signals released during cold ischemia storage activate NLRP3 inflammasome in myeloid cells and influence early allograft function in liver transplantation

doi: 10.1016/j.ebiom.2022.104419

Figure Lengend Snippet: DAMPs in eiOPS prime human primary monocytes. (a) Fold change of expression of IL6, TNFA, IL1B , and NLRP3 from human primary monocytes treated with LPS (500 ng/mL) or eiOPS for 16 h as detected by RT-qPCR. n = 4. (b–d) Representative cropped Western blots of intracellular pro-IL-1β (Santa Cruz Biotechnologies Cat# sc-7884, RRID: AB_2124476 ) and β-actin (Santa Cruz Biotechnologies Cat# Sc-47778 HRP, RRID: AB_2714189 ) from human primary monocytes primed for 16 h with eiOPS from different donors (b); different concentrations of LPS (c); and eiOPS in the presence of cytochalasin D (10 μM), MDC (250 μM), IL-18BP (100 ng/mL), anti-HMGB1 (100 ng/mL), anti-TLR2 (100 ng/mL), and mouse IgG isotype control (100 ng/mL), respectively (d). Alternatively, eiOPS previously incubated with proteinase K (1 mg/mL) or DNase I (100 U/mL) was used, as well as TLR2 activator Pam3csK4 (1 μg/mL) (d). (e) Release of IL-1β from human primary monocytes primed with LPS or eiOPS for 16 h, then activated with nigericin for 2 h. n = 4. (f) Release of IL-1β from human primary monocytes primed for 4 h with LPS or Pam3csK4, then incubated with eiOPS for 16 h. n = 4. Representative images are shown from 3 independent experiments. Results are presented as mean ± SEM; each sample was tested in duplicate; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001 [one-way ANOVA followed by Bonferroni test (a) or unpaired Student's t test (e, f)].

Article Snippet: The human macrophage THP-1 cell line was from the American Type Culture Collection (Cat# TIB-202, RRID: CVCL_0006).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation