Journal: Molecules

Article Title: ISOC1 Modulates Inflammatory Responses in Macrophages through the AKT1/PEX11B/Peroxisome Pathway

doi: 10.3390/molecules27185896

Figure Lengend Snippet: After LPS treatment, peroxisome level decreases more significantly in ISOC1-deficient cells than in wild-type cells. Wild-type or ISOC1-deficient RAW264.7 cells were treated with LPS (300 ng/mL) for 24 h. ( A ) Peroxisomes were labeled with ABCD3 and were observed with fluorescence microscopy. Scale bar = 10 μm. Mean gray values were analyzed by Image J v1.51 (Wayne Rasband, Bethesda, MD, USA). ( B ) Cell lysates were subjected to WB with antibodies to ABCD3, PEX11B, and PEX1. ( C ) O2—scavenging capacity was measured using a superoxide anion scavenging capacity assay Kit. ( D ) The content of 8-OHDG was analyzed by an oxidative RNA Damage ELISA Kit. All data are shown as mean ± s.e.m ( n = 3). * p < 0.05, ** p < 0.01; n.s. not significant. Two-way ANOVA followed by Bonferroni post hoc test was used for data analysis.

Article Snippet: The primary antibodies used included 1/1000 anti-α-Tubulin (AF0001), 1/1000 anti-pan Phospho-Serine/Threonine (anti-Pan-P) (AF5725), 1/500 anti-HA (AF5057) (Beyotime), 1/1000 anti-Isoc1 (PA5-67096), anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744) (ThermoFisher Scientific, San Jose, CA, USA), 1/1000 anti-COX-2 (12282), 1/1000 anti-AKT (4691), 1/1000 anti-phospho-AKT (4060), 1/500 anti-IκBα (4812), 1/500 anti-phospho-IκBα (9246), 1/500 anti-extracellular signal-regulated kinase (ERK1/2) (4696), 1/200 anti-phospho-ERK1/2 (4370)(Cell Signaling Technology, Inc., Danvers, MA, USA), 1/500 anti-ATP-binding cassette sub-family D member 3 (ABCD3) (sc-514728) (ABCD3), 1/500 anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744), and 1/500 anti-Ubiquitin (sc-271289) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Labeling, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Molecules

Article Title: ISOC1 Modulates Inflammatory Responses in Macrophages through the AKT1/PEX11B/Peroxisome Pathway

doi: 10.3390/molecules27185896

Figure Lengend Snippet: ISOC1 affects peroxisomes through the AKT1 pathway but not the NF-κB pathway. Wild-type or ISOC1-deficient RAW264.7 were pretreated with 10 μM Perifosine for 12 h or 10 μM BAY 11-7082 for 1.5 h, stimulated with 300 ng/mL LPS for 24 h. ( A , E ) Peroxisomes were labeled with ABCD3 and were observed with fluorescence microscopy. Scale bar = 10 μm. Mean gray value were analyzed by Image J. ( B , F ) WB was used to detect the expression levels of ABCD3, PEX11B, and PEX1. ( C , G ) O2− scavenging capacity was measured using a superoxide anion scavenging capacity assay Kit. ( D , H ) The content of 8-OHDG was analyzed by an oxidative RNA Damage ELISA Kit. All data are shown as mean ± s.e.m ( n = 3). * p < 0.05, ** p < 0.01, n.s. not significant. Two-way ANOVA followed by Bonferroni post hoc test was used for data analysis.

Article Snippet: The primary antibodies used included 1/1000 anti-α-Tubulin (AF0001), 1/1000 anti-pan Phospho-Serine/Threonine (anti-Pan-P) (AF5725), 1/500 anti-HA (AF5057) (Beyotime), 1/1000 anti-Isoc1 (PA5-67096), anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744) (ThermoFisher Scientific, San Jose, CA, USA), 1/1000 anti-COX-2 (12282), 1/1000 anti-AKT (4691), 1/1000 anti-phospho-AKT (4060), 1/500 anti-IκBα (4812), 1/500 anti-phospho-IκBα (9246), 1/500 anti-extracellular signal-regulated kinase (ERK1/2) (4696), 1/200 anti-phospho-ERK1/2 (4370)(Cell Signaling Technology, Inc., Danvers, MA, USA), 1/500 anti-ATP-binding cassette sub-family D member 3 (ABCD3) (sc-514728) (ABCD3), 1/500 anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744), and 1/500 anti-Ubiquitin (sc-271289) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Labeling, Fluorescence, Microscopy, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecules

Article Title: ISOC1 Modulates Inflammatory Responses in Macrophages through the AKT1/PEX11B/Peroxisome Pathway

doi: 10.3390/molecules27185896

Figure Lengend Snippet: LPS induces the expression of ISOC1, and ISOC1-deficient macrophages show a higher inflammation with LPS treatment. ( A , B ) RAW264.7 cells were treated with specified concentration of LPS; ISOC1 expression levels were measured by qRT-PCR ( A ) and Western blotting ( B ). ( C ) The protein levels of ISOC1 were measured by WB in WT and ISOC1-deficient cell lines. ( D ) qRT-PCR was used to detect the expression levels of IL-1β, IFN-γ, IL-6, and IL-12 with 300 ng/mL LPS for 24 h. ( E ) Western blotting was used to detect COX-2 protein levels in wild-type and ISOC1-deficient RAW264.7 stimulated with 300 ng/mL LPS for 24 h. ( A , D ) were representative of one experiment with at least three independent biological replicates; a single data point represented one technical repeat. All data are shown as mean ± s.e.m. ( n = 3). * p < 0.05, ** p < 0.01; n.s. not significant. ANOVA followed by Bonferroni post hoc test was used for data analysis.

Article Snippet: The primary antibodies used included 1/1000 anti-α-Tubulin (AF0001), 1/1000 anti-pan Phospho-Serine/Threonine (anti-Pan-P) (AF5725), 1/500 anti-HA (AF5057) (Beyotime), 1/1000 anti-Isoc1 (PA5-67096), anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744) (ThermoFisher Scientific, San Jose, CA, USA), 1/1000 anti-COX-2 (12282), 1/1000 anti-AKT (4691), 1/1000 anti-phospho-AKT (4060), 1/500 anti-IκBα (4812), 1/500 anti-phospho-IκBα (9246), 1/500 anti-extracellular signal-regulated kinase (ERK1/2) (4696), 1/200 anti-phospho-ERK1/2 (4370)(Cell Signaling Technology, Inc., Danvers, MA, USA), 1/500 anti-ATP-binding cassette sub-family D member 3 (ABCD3) (sc-514728) (ABCD3), 1/500 anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744), and 1/500 anti-Ubiquitin (sc-271289) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Concentration Assay, Quantitative RT-PCR, Western Blot

Journal: Molecules

Article Title: ISOC1 Modulates Inflammatory Responses in Macrophages through the AKT1/PEX11B/Peroxisome Pathway

doi: 10.3390/molecules27185896

Figure Lengend Snippet: ISOC1 mediates macrophage inflammatory response via AKT1 and NF-κB pathway. ( A ) Wild-type or ISOC1-deficient RAW264.7 cells were stimulated with 300 ng/mL LPS for 24 h. Cell lysates were collected, and western blotting was used to detect the phosphorylation levels of AKT1, IκBα, and ERK. ( B , C ) ISOC1-deficient RAW264.7 cells were pretreated with 10 μM Perifosine for 12 h ( B ) or 10 μM BAY 11-7082 for 1.5 h ( C ) and stimulated with 300 ng/mL LPS for 24 h. Expressions of the COX-2 protein were detected by Western blotting. ( D , E ) Wild-type and ISOC1-deficient RAW264.7 cells were pretreated with 10 μM Perifosine for 12 h ( D ) or 10 μM BAY 11-7082 for 1.5 h ( E ) and stimulated with 300 ng/mL LPS for 24 h. qRT-PCR was used to detect the expression levels of IL-1β, IFN-γ, IL-6, and IL-12. All data are shown as mean ± s.e.m ( n = 3). * p < 0.05, ** p < 0.01; n.s. not significant. Two-way ANOVA followed by Bonferroni post hoc test was used for data analysis.

Article Snippet: The primary antibodies used included 1/1000 anti-α-Tubulin (AF0001), 1/1000 anti-pan Phospho-Serine/Threonine (anti-Pan-P) (AF5725), 1/500 anti-HA (AF5057) (Beyotime), 1/1000 anti-Isoc1 (PA5-67096), anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744) (ThermoFisher Scientific, San Jose, CA, USA), 1/1000 anti-COX-2 (12282), 1/1000 anti-AKT (4691), 1/1000 anti-phospho-AKT (4060), 1/500 anti-IκBα (4812), 1/500 anti-phospho-IκBα (9246), 1/500 anti-extracellular signal-regulated kinase (ERK1/2) (4696), 1/200 anti-phospho-ERK1/2 (4370)(Cell Signaling Technology, Inc., Danvers, MA, USA), 1/500 anti-ATP-binding cassette sub-family D member 3 (ABCD3) (sc-514728) (ABCD3), 1/500 anti-PEX11B (PA5-37011), 1/500 anti-PEX1 (PA5-100677), 1/500 anti-PEX3 (PA5-42814), 1/500 anti-PEX7 (PA5-101744), and 1/500 anti-Ubiquitin (sc-271289) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Quantitative RT-PCR, Expressing

Journal: American Journal of Cancer Research

Article Title: MCPB-21 targets glycogenin-2 to regulate fatty acid oxidation and promote ferroptosis of breast cancer

doi: 10.62347/DIDU5607

Figure Lengend Snippet: MCPB-21 inhibits fatty acid β-oxidation and induces ferroptosis in breast cancer cells of MDA-AB-231 and MCF-7. (A) Representative protein bands of ACOX1, ABCD3, ABCD4, EHHADH, and CPT1α in MDA-AB-231 and MCF-7. Relative expression statistics of ACOX1, ABCD3, ABCD4, EHHADH, and CPT1α in MDA-AB-231 (B) and MCF-7 (C). (D) Representative protein bands of GPX4, SLC7A11, and ACSL4 in MDA-AB-231 and MCF-7. Relative expression statistics of GPX4, SLC7A11, and ACSL4 in MDA-AB-231 (E) and MCF-7 (F). Compared with control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Following this, the membranes were incubated overnight at 4°C with primary antibodies: ABCD3 (ATP Binding Cassette Subfamily D Member 3, Huabio, HA721969, 1:2000, Hangzhou, China), ABCD4 (ATP Binding Cassette Subfamily D Member 4, Huabio, ER62551, 1:2000, Hangzhou, China), ACOX1 (Zenbio, R23371 , 1:1000, North Carolina, USA), ACSL4 (Acyl-CoA Synthetase Long Chain Family Member 4, Proteintech, 22401-1-AP, 1:2000, Wuhan, China), CPT1α (Carnitine palmitoyltransferase 1α, Bioss, BS-23779R, 1:2000, Beijin, China), EHHADH (Peroxisomal l-bifunctional enzyme, Cusabio, CSB-PA600104LA01HU, 1:2000, Wuhan, China), GPX4 (Abclonal, A1933, 1:15000, Wuhan, China), GYG2 (Proteintech, 13150-1-AP, 1:1000, Wuhan, China), SLC7A11 (Solute Carrier Family 7 Member 11, Abclonal, A2413, 1:1000, Wuhan, China), and β-actin (Abclonal, AC026, 1:50000, Wuhan, China).

Techniques: Expressing, Control

Journal: American Journal of Cancer Research

Article Title: MCPB-21 targets glycogenin-2 to regulate fatty acid oxidation and promote ferroptosis of breast cancer

doi: 10.62347/DIDU5607

Figure Lengend Snippet: MCPB-21 induces ferroptosis by regulating fatty acid β-oxidation through ACOX1. (A) Oil red staining results of MDA-AB-231 and MCF-7. (B) Statistical results of Oil red staining of MDA-AB-231 and MCF-7. (C) Representative images of ROS in MDA-AB-231 and MCF-7. (D) Statistical results of ROS in MDA-AB-231 and MCF-7. (E) Representative protein bands of ACOX1, ABCD3, ABCD4, EHHADH, and CPT1α in MDA-AB-231 and MCF-7. Relative expression statistics of ACOX1, ABCD3, ABCD4, EHHADH, and CPT1α in MDA-AB-231 (F) and MCF-7 (G). (H) Representative protein bands of GPX4, SLC7A11, and ACSL4 in MDA-AB-231 and MCF-7. Relative expression of GPX4, SLC7A11, and ACSL4 in MDA-AB-231 (I) and MCF-7 (J). Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001. Compared with MCPB-21, # P < 0.05, ## P < 0.01, and ### P < 0.001.

Article Snippet: Following this, the membranes were incubated overnight at 4°C with primary antibodies: ABCD3 (ATP Binding Cassette Subfamily D Member 3, Huabio, HA721969, 1:2000, Hangzhou, China), ABCD4 (ATP Binding Cassette Subfamily D Member 4, Huabio, ER62551, 1:2000, Hangzhou, China), ACOX1 (Zenbio, R23371 , 1:1000, North Carolina, USA), ACSL4 (Acyl-CoA Synthetase Long Chain Family Member 4, Proteintech, 22401-1-AP, 1:2000, Wuhan, China), CPT1α (Carnitine palmitoyltransferase 1α, Bioss, BS-23779R, 1:2000, Beijin, China), EHHADH (Peroxisomal l-bifunctional enzyme, Cusabio, CSB-PA600104LA01HU, 1:2000, Wuhan, China), GPX4 (Abclonal, A1933, 1:15000, Wuhan, China), GYG2 (Proteintech, 13150-1-AP, 1:1000, Wuhan, China), SLC7A11 (Solute Carrier Family 7 Member 11, Abclonal, A2413, 1:1000, Wuhan, China), and β-actin (Abclonal, AC026, 1:50000, Wuhan, China).

Techniques: Staining, Expressing, Control

Journal: American Journal of Cancer Research

Article Title: MCPB-21 targets glycogenin-2 to regulate fatty acid oxidation and promote ferroptosis of breast cancer

doi: 10.62347/DIDU5607

Figure Lengend Snippet: MCPB-21 regulates fatty acid oxidation and ferroptosis in breast cancer cells via GYG2. (A) Representative bands of GYG2 in MDA-AB-231 and MCF-7. (B) Relative expression statistics of GYG2. (C) Oil red staining results of MDA-AB-231 and MCF-7. (D) Oil red staining results of MDA-AB-231 and MCF-7. (E) ROS statistics of MDA-AB-231 and MCF-7. (F) Representative images of ROS in MDA-AB-231 and MCF-7. (G) Representative protein bands of ACOX1, ABCD3, ABCD4, EHHADH, and CPT1α in MDA-AB-231 and MCF-7. Relative expression statistics of ACOX1, ABCD3, ABCD4, EHHADH, and CPT1α in MDA-AB-231 (H) and MCF-7 (I). (J) Representative protein bands of GPX4, SLC7A11, and ACSL4 in MDA-AB-231 and MCF-7. Statistical results of relative expression of GPX4, SLC7A11, and ACSL4 in MDA-AB-231 (K) and MCF-7 (L). Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001. Compared with MCPB-21, # P < 0.05, ## P < 0.01, and ### P < 0.001.

Article Snippet: Following this, the membranes were incubated overnight at 4°C with primary antibodies: ABCD3 (ATP Binding Cassette Subfamily D Member 3, Huabio, HA721969, 1:2000, Hangzhou, China), ABCD4 (ATP Binding Cassette Subfamily D Member 4, Huabio, ER62551, 1:2000, Hangzhou, China), ACOX1 (Zenbio, R23371 , 1:1000, North Carolina, USA), ACSL4 (Acyl-CoA Synthetase Long Chain Family Member 4, Proteintech, 22401-1-AP, 1:2000, Wuhan, China), CPT1α (Carnitine palmitoyltransferase 1α, Bioss, BS-23779R, 1:2000, Beijin, China), EHHADH (Peroxisomal l-bifunctional enzyme, Cusabio, CSB-PA600104LA01HU, 1:2000, Wuhan, China), GPX4 (Abclonal, A1933, 1:15000, Wuhan, China), GYG2 (Proteintech, 13150-1-AP, 1:1000, Wuhan, China), SLC7A11 (Solute Carrier Family 7 Member 11, Abclonal, A2413, 1:1000, Wuhan, China), and β-actin (Abclonal, AC026, 1:50000, Wuhan, China).

Techniques: Expressing, Staining, Control

Journal: American Journal of Cancer Research

Article Title: MCPB-21 targets glycogenin-2 to regulate fatty acid oxidation and promote ferroptosis of breast cancer

doi: 10.62347/DIDU5607

Figure Lengend Snippet: MCPB-21 induces cell death in breast cancer cell lines MDA-MB-231 and MCF-7 via the ferroptosis pathway. (A, B) Flow cytometry analysis of cell death rates in each group, along with statistical analysis. (C, D) Detection of intracellular ROS levels in each group using the DCFH-DA fluorescent probe via flow cytometry, along with statistical analysis. (E) Representative Western blots for GPX4, SLC7A11, and ACSL4. Quantitative analysis of the expression levels of the aforementioned proteins in MDA-MB-231 (F) and MCF-7 (G) cells. Compared with the Control group: * P < 0.05, ** P < 0.001. Compared with the MCPB-21 group: ## P < 0.01, ### P < 0.001.

Article Snippet: Following this, the membranes were incubated overnight at 4°C with primary antibodies: ABCD3 (ATP Binding Cassette Subfamily D Member 3, Huabio, HA721969, 1:2000, Hangzhou, China), ABCD4 (ATP Binding Cassette Subfamily D Member 4, Huabio, ER62551, 1:2000, Hangzhou, China), ACOX1 (Zenbio, R23371 , 1:1000, North Carolina, USA), ACSL4 (Acyl-CoA Synthetase Long Chain Family Member 4, Proteintech, 22401-1-AP, 1:2000, Wuhan, China), CPT1α (Carnitine palmitoyltransferase 1α, Bioss, BS-23779R, 1:2000, Beijin, China), EHHADH (Peroxisomal l-bifunctional enzyme, Cusabio, CSB-PA600104LA01HU, 1:2000, Wuhan, China), GPX4 (Abclonal, A1933, 1:15000, Wuhan, China), GYG2 (Proteintech, 13150-1-AP, 1:1000, Wuhan, China), SLC7A11 (Solute Carrier Family 7 Member 11, Abclonal, A2413, 1:1000, Wuhan, China), and β-actin (Abclonal, AC026, 1:50000, Wuhan, China).

Techniques: Flow Cytometry, Western Blot, Expressing, Control

Journal: American Journal of Cancer Research

Article Title: MCPB-21 targets glycogenin-2 to regulate fatty acid oxidation and promote ferroptosis of breast cancer

doi: 10.62347/DIDU5607

Figure Lengend Snippet: Effects of CBD and MCPB-21 on the formation of transplant tumors in MDA-AB-231. A. Mouse transplantation tumor. B. Weight of transplant tumor. C. The values of xenografted tumor volumes. D. H&E staining. E. Representative immunohistochemistry images of Ki67, GPX4, SLC7A11, and ACSL4. F. Statistical results of Ki67. G. Statistical results of GPX4, SLC7A11, and ACSL4 expression. H. Representative bands of GYG2, ACOX1, ABCD3, ABCD4, EHHADH, and CPT1a. I. Relative expression of GYG2. J. Relative expression of ACOX1, ABCD3, ABCD4, EHHADH, and CPT1a. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Following this, the membranes were incubated overnight at 4°C with primary antibodies: ABCD3 (ATP Binding Cassette Subfamily D Member 3, Huabio, HA721969, 1:2000, Hangzhou, China), ABCD4 (ATP Binding Cassette Subfamily D Member 4, Huabio, ER62551, 1:2000, Hangzhou, China), ACOX1 (Zenbio, R23371 , 1:1000, North Carolina, USA), ACSL4 (Acyl-CoA Synthetase Long Chain Family Member 4, Proteintech, 22401-1-AP, 1:2000, Wuhan, China), CPT1α (Carnitine palmitoyltransferase 1α, Bioss, BS-23779R, 1:2000, Beijin, China), EHHADH (Peroxisomal l-bifunctional enzyme, Cusabio, CSB-PA600104LA01HU, 1:2000, Wuhan, China), GPX4 (Abclonal, A1933, 1:15000, Wuhan, China), GYG2 (Proteintech, 13150-1-AP, 1:1000, Wuhan, China), SLC7A11 (Solute Carrier Family 7 Member 11, Abclonal, A2413, 1:1000, Wuhan, China), and β-actin (Abclonal, AC026, 1:50000, Wuhan, China).

Techniques: Transplantation Assay, Staining, Immunohistochemistry, Expressing, Control