Journal: Marine drugs

Article Title: Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells.

doi: 10.3390/md22010026

Figure Lengend Snippet: Figure 6. Target genes and the key protein identified from composite treatment (n = 3 in each group). (A) Venn diagram analysis indicated the number of genes of overlapping groups. In total, 411 common genes were further used for PPI analysis. (B) PPI analysis indicated EGR1 as the key regulatory protein.

Article Snippet: The antibodies for early growth response protein 1 (EGR1) (catalogue number: 4154S), growth arrest, and the DNA damage-inducible protein alpha (GADD45A) (catalogue number: 4632S), and β-actin (catalogue number: 4970S) were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques:

Journal: Marine drugs

Article Title: Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells.

doi: 10.3390/md22010026

Figure Lengend Snippet: Figure 7. The effect of chitotriose on the transcription and expression levels of Egr1 and Gadd45a (n = 3 in each group) with siRNA transfection. The mRNA level of (A) Egr1 and (B) Gadd45a detected via RT-qPCR. Asterisks (***) indicate significant differences at p-value < 0.001 compared with the CTL group. Sharps (#), (##), and (###) indicate significant differences at p-values < 0.05, 0.01 and 0.001 for each group before and after siRNA transfection. (C) Protein levels detected using the Western blot assay. (D) Cell viability detected via the CCK-8 assay. Sharps (###) indicate significant differences at p-value < 0.001 between the combined and DOX groups.

Article Snippet: The antibodies for early growth response protein 1 (EGR1) (catalogue number: 4154S), growth arrest, and the DNA damage-inducible protein alpha (GADD45A) (catalogue number: 4632S), and β-actin (catalogue number: 4970S) were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay

Journal: Genomics

Article Title: EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis.

doi: 10.1016/j.ygeno.2023.110683

Figure Lengend Snippet: Fig. 1. EGR1 expression is low in CEPs from IVDD patients. (A) GSE15227 analysis of DEGs in normal and degenerated IVD tissues; (B) The intersection between DEGs in Fig. 1A analysis and ferroptosis-related genes in the FerrDb database; (C–D) qRT-PCR or western blot detections of EGR1 mRNA and protein expression in CEP tissues from IVDD patients. Data were presented as mean ± standard deviation. Clinical experiment: IVDD, N = 20; Health, N = 8. *P < 0.05 versus the health group. EGR1, early growth response gene 1; IVDD, intervertebral disc degeneration; GSE, gene expression omnibus series; CEP, cartilage endplate; DEG, differentially expressed gene; qRT-PCR, quantitative reverse transcription- polymerase chain reaction; mRNA, messenger RNA.

Article Snippet: Methanal was utilized to fix cells for 10 min for DNA-protein crosslinking and an ultrasonic cell disruptor was adopted to fragment chromatin under the following conditions: 15 cycles of 10-s ultrasound at an interval of 10 s. Subsequently, samples were subjected to 10-min centrifugation (4 ◦C, 12000 g) and collected into 2 tubes where NC antibody Immunoglobulin G (IgG, ab172730, 1:100, Abcam) and specific antibody against target protein anti-EGR1 (4154S, 1:50, CST) were respectively added and incubated overnight at 4 ◦C for complete binding.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Gene Expression, Reverse Transcription, Polymerase Chain Reaction

Journal: Genomics

Article Title: EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis.

doi: 10.1016/j.ygeno.2023.110683

Figure Lengend Snippet: Fig. 2. EGR1 knockdown reduces IVD cartilage degeneration by repressing ferroptosis. (A-B) qRT-PCR and western blot examinations of EGR1 mRNA and protein expression before or after the transfection of sh-EGR1 in ICMT-treated CEP cells, respectively; (C) Kit detections of LDH levels in ICMT-treated CEP cells; (D) Flow cytometry observations of cell apoptosis in ICMT-treated CEP cells; (E) Western blot testing of expression levels of degeneration-related proteins (Col II, Aggrecan, MMP13, and ADAMTS-5) in ICMT-treated CEP cells; (F–H) Kit detections of contents of iron, MDA, and GSH in ICMT-treated CEP cells; (I) DCFH-DA molecular probe measurement of ROS expression in ICMT-treated CEP cells; (J) Transmission electron microscope observations of mitochondrial ultrastructure changes in ICMT-treated CEP cells; (K) Western blot examinations of levels of ferroptosis-related proteins (TFR1, ACSL4, GPX4, and SLC7A11) in ICMT-treated CEP cells. Data were presented as mean ± standard deviation. Each experiment was repeated 3 times. *P < 0.05 versus the control group; #P < 0.05 versus the sh-NC group; &P < 0.05 versus the sh-EGR1 group. EGR1, early growth response gene 1; IVD, intervertebral disc; CEP, cartilage endplate; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; mRNA, messenger RNA; LDH, lactic dehydrogenase; MDA, malondial dehyde; GSH, glutathione; DCFH-DA, 20, 70-dichlorofluorescein diacetate; ROS, reactive oxygen species; Sh, short hairpin; NC, negative control.

Article Snippet: Methanal was utilized to fix cells for 10 min for DNA-protein crosslinking and an ultrasonic cell disruptor was adopted to fragment chromatin under the following conditions: 15 cycles of 10-s ultrasound at an interval of 10 s. Subsequently, samples were subjected to 10-min centrifugation (4 ◦C, 12000 g) and collected into 2 tubes where NC antibody Immunoglobulin G (IgG, ab172730, 1:100, Abcam) and specific antibody against target protein anti-EGR1 (4154S, 1:50, CST) were respectively added and incubated overnight at 4 ◦C for complete binding.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Flow Cytometry, Transmission Assay, Microscopy, Standard Deviation, Control, Reverse Transcription, Polymerase Chain Reaction, Negative Control

Journal: Genomics

Article Title: EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis.

doi: 10.1016/j.ygeno.2023.110683

Figure Lengend Snippet: Fig. 3. EGR1 binds to the MAP3K14 promoter and facilitates MAP3K14 transcription. (A) Bioinformatic analysis to screen out the downstream target genes of EGR1; (B) Jaspar website analysis of binding between EGR1 and MAP3K14 promoter sequence; (C–F) qRT-PCR and western blot to determine MAP3K14 mRNA and protein expression in CEP tissues from IVDD patients and ICMT-treated CEP cells; (G- H) Dual-luciferase reporter gene and ChIP assay to validate the binding relationship between EGR1 and MAP3K14; (I-J) qRT-PCR or western blot to test mRNA and protein expression levels of MAP3K14 in ICMT-treated CEP cells. Data were presented as mean ± standard deviation. Clinical experiment: IVDD, N = 20; Health, N = 8. Cell experiment was repeated 3 times. *P < 0.05 versus the health, control, IgG, or sh-NC + oe-NC group; #P < 0.05 versus the sh-NC group; &P < 0.05 versus the sh-EGR1 + oe-NC group. EGR1, early growth response gene 1; MAP3K14, mitogen-activated protein kinase kinase kinase 14; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; mRNA, messenger RNA; CEP, cartilage endplate; IVDD, intervertebral disc degeneration; ChIP, chromatin immunopre cipitation; Sh, short hairpin; NC, negative control; Oe, overexpression; IgG, Immunoglobulin G.

Article Snippet: Methanal was utilized to fix cells for 10 min for DNA-protein crosslinking and an ultrasonic cell disruptor was adopted to fragment chromatin under the following conditions: 15 cycles of 10-s ultrasound at an interval of 10 s. Subsequently, samples were subjected to 10-min centrifugation (4 ◦C, 12000 g) and collected into 2 tubes where NC antibody Immunoglobulin G (IgG, ab172730, 1:100, Abcam) and specific antibody against target protein anti-EGR1 (4154S, 1:50, CST) were respectively added and incubated overnight at 4 ◦C for complete binding.

Techniques: Binding Assay, Sequencing, Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Standard Deviation, Control, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Over Expression

Journal: Genomics

Article Title: EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis.

doi: 10.1016/j.ygeno.2023.110683

Figure Lengend Snippet: Fig. 4. EGR1 elevates the transcription of MAP3K14 and activates the NF-κB pathway, thereby promoting ferroptosis and aggravating IVD cartilage degeneration. (A-C) Western blot to measure the expression of NF-κB p65 in GEP tissues from IVDD patients and ICMT-treated CEP cells; (D) Kits to detect LDH levels in ICMT- treated CEP cells; (E) Flow cytometry to determine cell apoptosis in ICMT-treated CEP cells; (F) Western blot to detect expression levels of degeneration-related proteins (Col II, Aggrecan, MMP13, and ADAMTS-5) in ICMT-treated CEP cells; (G-I) Kits to determine iron contents and MDA and GSH levels in ICMT-treated CEP cells; (J) DCFH-DA molecular probes to test ROS expression in ICMT-treated CEP cells; (K) Transmission electron microscope to observe ultrastructural changes of mitochondria in ICMT-treated CEP cells; (L) Western blot to measure expression levels of ferroptosis-related proteins (TFR1, ACSL4, GPX4, and SLC7A11) in ICMT-treated CEP cells. Data were presented as mean ± standard deviation. Clinical experiment: IVDD, N = 20; Health, N = 8. Cell experiment was repeated 3 times. *P < 0.05 versus the health, control, or sh-NC + oe-NC group; #P < 0.05 versus the sh-EGR1 + oe-NC group; &P < 0.05 versus the sh-EGR1 + oe-MAP3K14 group. EGR1, early growth response gene 1; MAP3K14, mitogen-activated protein kinase kinase kinase 14; NF-κB, nuclear factor-κB; IVD, intervertebral disc; CEP, cartilage endplate; IVDD, intervertebral disc degeneration; LDH, lactic dehydrogenase; MDA, malondialdehyde; GSH, glutathione; DCFH-DA, 20, 70-dichlorofluor escein diacetate; ROS, reactive oxygen species; Sh, short hairpin; NC, negative control; Oe, overexpression.

Article Snippet: Methanal was utilized to fix cells for 10 min for DNA-protein crosslinking and an ultrasonic cell disruptor was adopted to fragment chromatin under the following conditions: 15 cycles of 10-s ultrasound at an interval of 10 s. Subsequently, samples were subjected to 10-min centrifugation (4 ◦C, 12000 g) and collected into 2 tubes where NC antibody Immunoglobulin G (IgG, ab172730, 1:100, Abcam) and specific antibody against target protein anti-EGR1 (4154S, 1:50, CST) were respectively added and incubated overnight at 4 ◦C for complete binding.

Techniques: Western Blot, Expressing, Flow Cytometry, Transmission Assay, Microscopy, Standard Deviation, Control, Negative Control, Over Expression

Journal: Genomics

Article Title: EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis.

doi: 10.1016/j.ygeno.2023.110683

Figure Lengend Snippet: Fig. 5. EGR1 silencing curtails ferroptosis to ameliorate CEP degeneration in IVDD mice by inactivating the MAP3K14/NF-κB axis. (A) HE staining to assess pathological change in the cartilage of IVDD mice; (B) Safranin O-fast green staining to monitor the changes of CEP in IVDD mice; (C–D) qRT-PCR or western blot to assess expression levels of ERG1, MAP3K14, and NF-κB p65 in CEP tissues of IVDD mice; (E) Western blot to test expression levels of degeneration–related proteins (Col II, Aggrecan, MMP13, and ADAMTS-5); (F–I) Kits to determine iron content and ROS, MDA, and GSH levels in CEP tissues of IVDD mice; (J) Western blot to detect expression levels of ferroptosis-related proteins (TFR1, ACSL4, GPX4, and SLC7A11). Data were presented as mean ± standard deviation. Animal experiment: N = 6. *P < 0.05 versus the sham group; #P < 0.05 versus the sh-NC group. EGR1, early growth response gene 1; MAP3K14, mitogen- activated protein kinase kinase kinase 14; CEP, cartilage endplate; IVDD, intervertebral disc degeneration; NF-κB, nuclear factor-κB; HE, hematoxylin-eosin; qRT- PCR, quantitative reverse transcription-polymerase chain reaction; MDA, malondialdehyde; GSH, glutathione; ROS, reactive oxygen species; Sh, short hairpin; NC, negative control; Oe, overexpression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Methanal was utilized to fix cells for 10 min for DNA-protein crosslinking and an ultrasonic cell disruptor was adopted to fragment chromatin under the following conditions: 15 cycles of 10-s ultrasound at an interval of 10 s. Subsequently, samples were subjected to 10-min centrifugation (4 ◦C, 12000 g) and collected into 2 tubes where NC antibody Immunoglobulin G (IgG, ab172730, 1:100, Abcam) and specific antibody against target protein anti-EGR1 (4154S, 1:50, CST) were respectively added and incubated overnight at 4 ◦C for complete binding.

Techniques: Staining, Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Over Expression