Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: LOX-1 inhibition reduces ROS generation, autophagy, mtDNA damage, and NLRP3 inflammasome expression. (A) LOX-1 inhibition by pre-treatment with a specific antibody or siRNA transfection blocks LOX-1 expression. (B) LOX-1 inhibition reduces cellular ROS generation. (C) LOX-1 inhibition decreases autophagic flux. (D and E) LOX-1 inhibition protects mtDNA from damage and inhibits NLRP3 expression. Cells were pre-treated with 10 μg/mL of LOX-1 antibody (Ab) or transfected with 20 nM of siRNA directed at LOX-1 for 24 h; then the cells were treated with 10 ng/mL of LPS for another 24 h. ssc, scrambled siRNA control. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Inhibition, Expressing, Transfection, Control

Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: DNase II knockdown enhances the expression of LOX-1 and NLRP3 inflammasome, and aggravates mtDNA damage. (A) Expression of DNase II is inhibited by siRNA transfection. (C–E) DNase II knockdown enhances ROS generation, mtDNA damage, autophagy, and expression of NLRP3. Cells were transfected with 20 nM DNase II siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. ssc, scrambled siRNA control. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Knockdown, Expressing, Transfection, Control

Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: Study in mice primary peritoneal macrophages confirms the results in THP-1 cells. (A and B) LOX-1 knockdown inhibits mtDNA damage and expression LC3-II, P62, and NLRP3 inflammasome expression. Cells were transfected with LOX-1 siRNA for 24 h, then the cells were treated with LPS for another 24 h. (C to E) Both ROS inhibitors apocynin and YCG063 decrease mtROS generation, mtDNA damage, as well as expression of LC3-II, P62, LOX-1, and NLRP3 inflammasome expression. Cells were treated with LPS for 24 h in the presence or absence of 0.01 mM apocynin and 10 µM YCG063. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05. (F) Proposed signalling pathway linking LOX-1 to mtDNA damage, autophagy, and NLRP3 activation. There appears to be a positive feedback loop between LOX-1 and ROS. Activation of both LOX-1 and ROS induces mtDNA damage. Though most of damaged DNA can be removed by autophagy and DNase II degradation, some damaged mtDNA that persists may result in activation of NLRP3 inflammasome.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Knockdown, Expressing, Transfection, Activation Assay

Journal: Cardiovascular Research

Article Title: LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

doi: 10.1093/cvr/cvu114

Figure Lengend Snippet: LPS and mtROS generation. (A) LPS induces mtROS generation in a dose-dependent manner. Cells were treated with 1–100 ng/mL of LPS for 24 h before measurement of mtROS. (B) LOX-1 knockdown by antibody or siRNA transfection inhibits mtROS generation. Cells were pre-treated with 10 μg/mL of LOX-1 antibody (Ab) or transfected with 20 nM its siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. (C) ROS inhibitors apocynin and YCG063 inhibit mtROS generation. Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of 1 mM apocynin and 10 µM YCG063. (D) Autophagy inducer rapamycin inhibits, while autophagy inhibitor 3-methyladenine enhances mtROS generation. Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of 10 nM rapamycin and 5 mM 3-methyladenine. (E) DNase II knockdown by its siRNA transfection increases mtROS generation. Cells were transfected with 20 nM DNase II siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Article Snippet: Briefly, cells were treated with the siRNA duplex solution for 24 h. The medium was then replaced with normal culture medium, and cells were treated with 10 ng/mL of LPS for another 24 h. As control, cells were transfected with sequence scrambled siRNA control (ssc, Santa Cruz).

Techniques: Knockdown, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Four-octyl itaconate activates Keap1-Nrf2 signaling to protect neuronal cells from hydrogen peroxide

doi: 10.1186/s12964-018-0294-2

Figure Lengend Snippet: Nrf2 activation mediates 4-octyl itaconate-induced neuronal cell protection against H 2 O 2 . SH-SY5Y cells ( a - e ) or the primary murine neurons ( i - k ), with the applied Nrf2 shRNA or the scramble control shRNA (“shC”), were either untreated or treated with 4-octyl itaconate (OI), mRNA and protein expression of listed genes were shown ( a - c , and i ); Cells were pretreated for 30 min with OI (25 μM), followed by stimulation of H 2 O 2 (300 μM) for indicated time, cell viability (CCK-8 OD, d ), cell death (LDH release, j ) and apoptosis (TUNEL ratio increase, e , and k ) were tested. Stable SH-SY5Y cells, with the CRISPR/Cas9-Nrf2 KO construct (“Nrf2-KO”) or the CRISPR/Cas9 control construct (“Cas9-c”), were treated with 4-octyl itaconate (OI), listed proteins were shown ( f ); Cells were pretreated for 30 min with OI (25 μM), followed by stimulation of H 2 O 2 (300 μM) for indicated time, cell viability ( g ) and apoptosis ( h ) were tested. Expression of listed proteins were quantified and normalized to the loading control ( c , f and i ). “shNrf2 (m)” stands for murine Nrf2 shRNA ( I - K ). Bars stand for mean ± standard deviation (S.D., n = 5). # P < 0.05 vs. “shC” cells ( a , b , d and e ). # P < 0.05 ( g , h , j and k )

Article Snippet: Two different lentivirus-packed Nrf2 shRNAs, targeting non-overlapping sequence of human Nrf2 (sc-37030-V/“shNrf2–1” and sc-44332-V/“shNrf2–2”) , as well as the lentiviral murine Nrf2 shRNA [sc-37049-V, “shNrf2 (m)”] and the scramble nonsense control shRNA (“shC”, sc-108080) were purchased from Santa Cruz Biotech (Santa Cruz, CA). shRNA lentivirus were added to cultured cells in the presence of polybrene (5 μg/mL) for 48 h. Puromycin (1.0 μg/mL) was then included to select stable cells for 4–5 passages.

Techniques: Activation Assay, shRNA, Control, Expressing, CCK-8 Assay, TUNEL Assay, CRISPR, Construct, Standard Deviation

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Western Blotting and chromosome aberration analysis. ( a ) Western Blotting analysis for protein expression; ( b ) Frequencies of chromatid breaks 48 hr after transfection with siRNA against Bmi-1 and scramble RNA (siControl). Two hundred metaphases were analyzed. Error bars indicate standard deviation (SD) and ( c ) Examples of chromatid breaks (indicated by arrows) in HeLa cells.

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Transfection, Standard Deviation

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Bmi-1 knockdown-induced chromatid breaks were rescued by knockdown of cyclin E. ( a ) Western Blotting analysis confirmed the effective knockdown of cylin E and ( b ) Frequencies of chromatid breaks 48 hr after transfection with siRNA against Bmi-1 together with siRNA against cyclin E or scramble RNA. Two hundred metaphases were analyzed. Error bars indicate SD.

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Western Blot, Transfection

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Bmi-1 knockdown-induced chromatid breaks were rescued by ectopic p21 overexpression. ( a ) Western Blotting analysis confirmed the effective p21 overexpression and ( b ) Frequencies of chromatid breaks 48 hr after transfection with siRNA against Bmi-1 together with pCEP-p21 or empty vector. Two hundred metaphases were analyzed. Error bars indicate SD.

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Over Expression, Western Blot, Transfection, Plasmid Preparation

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

doi: 10.1002/ijc.29114

Figure Lengend Snippet: Bmi-1 knockdown increased the proportions of cells with DNA synthesis and replicative stress. ( a ) Examples of BrdU-staining to indicate the cells with DNA synthesis; ( b ) Frequencies of BrdU-positive cells after transfection with siRNA against Bmi-1 or scramble RNA; 500 cells were analyzed and error bars indicate SD; (c) Example of pan-nuclear staining of γ-H2AX (indicated by arrows) and ( d ) Frequencies of pan-nuclear γ-H2AX-positive cells after transfection with scramble siRNA, siRNA against Bmi-1, the combination of siRNA against Bmi-1 and empty vectors, or the combination of siRNA against Bmi-1 and p21 overexpression vectors. Five hundred cells were analyzed and error bars indicate SD. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com .]

Article Snippet: Small interfering RNAs (siRNA) against human Bmi-1, cylin E or control siRNA (scramble RNA sequence) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, DNA Synthesis, BrdU Staining, Transfection, Staining, Over Expression