Journal: Journal of Cellular and Molecular Medicine

Article Title: Therapeutic potential of melatonin in the intervertebral disc degeneration through inhibiting the ferroptosis of nucleus pulpous cells

doi: 10.1111/jcmm.17818

Figure Lengend Snippet: The list of primers was utilized for qPCR.

Article Snippet: The following primary polyclonal antibodies were used: rabbit anti‐mouse IL‐6 (Cat. No. PAA079Mu01), rabbit anti‐mouse IL‐1β (Cat. No. PAA563Mu01), rabbit anti‐mouse TNF‐α (Cat. No. PAA133Mu01), rabbit anti‐mouse iNOS (Cat. No. PAA837Mu01), rabbit anti‐mouse COX2 (Cat. No. PAA699Mu01), rabbit anti‐mouse ACAN (Cat. No. PAB908Mu01), rabbit anti‐mouse COL2A1 (Cat. No. PAD194Mu01), rabbit anti‐mouse MMP‐13 (Cat. No. PAA099Mu01), rabbit anti‐mouse GPX4 (Cat. No. PAC994Mu01), rabbit anti‐mouse LPCAT3 (Cat. No. PAG531Mu01) that from the Cloud Clone Corporation, Wuhan, China; rabbit anti‐mouse ADAMTS‐4 (Cat. No. ab185722), rabbit anti‐mouse ADAMTS‐5 (Cat. No. ab41037) that from the Abcam, Cambridge, England; rabbit anti‐mouse anti‐ACSL4 (Cat. No. ab2844946) that from the Affbiotech, Jiangsu, China; and rabbit anti‐mouse SLC7A11 (Cat. No. BM5318) that from the Boster Biological Technology Co., Ltd, Wuhan, China.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Ferroptosis is controlled by the coordinated transcriptional regulation of glutathione and labile iron metabolism by the transcription factor BACH1

doi: 10.1074/jbc.ra119.009548

Figure Lengend Snippet: Figure 3. BACH1 decreases GSH by repressing the transcription of Slc7a11, Gclm, and Gclc. (A) Intracellular concentration of GSH in WT and Bach1-/- MEFs (7th, 9th, and 11th passage : P7, P9, and P11) by UHPLC/MS/MS. (B) ChIP-seq analysis of the binding of BACH1, MAFK for gene regions of Slc7a11, Gclm, and Gclc in M1 cells. (C) qPCR analysis for Slc7a11, Gclm, and Gclc mRNA relative to Actb mRNA in WT and Bach1-/- MEFs (P7, P9, P11). n = 3 of independent lots of MEFs per genotype. (D, E) Western blotting for BACH1, SLC7A11, GCLM, GCLC, and GAPDH in WT and Bach1-/- MEFs (P10) exposed to 25 µM MG132. Representative image (D) and the intensity of bands (E). The box and whisker plots of (A) show the 25th and 75th percentile quartiles and median values (center black line) and maximum and minimum values of the data. Error bars of (C, E) represent standard deviation. P-value of (A,C, and E) by t-test.

Article Snippet: The antibody for Slc7a11 (119-11215) was purchased by RayBiotech (Norcross, GA, USA).

Techniques: Concentration Assay, Tandem Mass Spectroscopy, ChIP-sequencing, Binding Assay, Western Blot, Whisker Assay, Standard Deviation

Journal: Cancers

Article Title: SHARPIN Enhances Ferroptosis in Synovial Sarcoma Cells via NF-κB- and PRMT5-Mediated PGC1α Reduction.

doi: 10.3390/cancers15133484

Figure Lengend Snippet: Figure 4. SHARPIN enhances the sensitivity of synovial sarcoma cell lines to ferroptosis via the PGC1α/SLC7A11 axis. (A–D) Viability assays of Aska (A,B) and Yamato (C,D) cells expressing scrambled or SHARPIN-specific shRNAs and treated with the indicated concentration of RSL3 (A,C) or erastin (B,D) for 24 h. (E) Immunoblot analyses of the effects of knockdown of SHARPIN on the expression levels of PGC1α, SLC7A11, SHARPIN, complex I, III, V, VDAC1/3, Parkin, BNIP3L/NIX, and LC3B in Yamato and Aska cells. (F) A qPCR analysis of the effect of transient SMART- pool siRNA-mediated knockdown of SHARPIN on SLC7A11 mRNA expression in Yamato cells. (G) Immunoblot analyses of the effects of knockdown of SHARPIN on the expression levels of NRF2 in Yamato cells. (H) Complex I activity in Yamato cells expressing scrambled or SHARPIN-specific shRNAs. Cells were seeded in identical numbers and incubated overnight. Signal intensity was then measured at the indicated time points. (I) ROS assay of Yamato cells expressing scrambled or SHARPIN-specific shRNAs. The cells were treated with or without 0.01 µM RSL3 for 24 h prior to the measurement of ROS activity. (J) GSH/GSSG ratio assay of Yamato cells expressing scrambled or SHARPIN-specific shRNAs. (K) Analysis of the relationship between SHARPIN mRNA expression levels and the GPX4 dependency of a bone and soft tissue sarcoma cohort using Chronos, a dynamic model of CRISPR data (CCLE database). The population below the first quantile (n = 18) was regarded as the low group, the population between the first and third quantile (n = 33) was regarded as the middle group, and the population above the third quantile (n = 18) was regarded as the high group. (A–D,F) Quantitative data are presented as the mean ± SD (n = 3). (K) A box-and-whisker plot is shown. (A–D,I,J) Statistical significance was calculated using one- or two-way ANOVA. * p < 0.05; ** p < 0.005; *** p < 0.0005; NS, not significant. (F) Statistical significance was calculated using a

Article Snippet: Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07- 412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology).

Techniques: Expressing, Concentration Assay, Western Blot, Knockdown, Activity Assay, Incubation, ROS Assay, CRISPR, Whisker Assay

Journal: Cancers

Article Title: SHARPIN Enhances Ferroptosis in Synovial Sarcoma Cells via NF-κB- and PRMT5-Mediated PGC1α Reduction.

doi: 10.3390/cancers15133484

Figure Lengend Snippet: Figure 5. Aberrant PGC1α expression overwhelms the regulatory effect of SHARPIN inhibition on ferroptosis in CCS. (A) A qPCR analysis of PGC1α mRNA expression in several sarcoma or non-sarcoma cell lines including CCS cell lines (SU and KAS). (B) Immunoblot analyses of SHARPIN, PRMT5, SDMA, SOX10, MITF, PGC1α and SLC7A11 in four permanent CCS cell lines, one primary CCS cell line, and HDF. (C) A qPCR analysis of SHARPIN mRNA expression in CCS clinical samples (n = 11) and normal tissues (n = 4). (D) The effect of knockdown of SHARPIN on PGC1α protein expression in SU and KAS cell lines. (E) Viability assays of SU and KAS cells expressing scrambled or SHARPIN-specific shRNAs. The cells were treated with or without the indicated concentration of RSL3 for 24 h. Cell viability was measured using the ratio of live cells in treated/control. (C) A box-and-whisker plot is shown. Statistical significance was calculated using a Mann–Whitney U test. * p < 0.05. (E) Statistical significance was calculated via a one-way ANOVA or Student’s t-test. Quantitative data are presented as the mean ± SD (n = 3). NS, not significant. The uncropped blots are shown in File S1.

Article Snippet: Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07- 412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology).

Techniques: Expressing, Inhibition, Western Blot, Knockdown, Concentration Assay, Control, Whisker Assay, MANN-WHITNEY

Journal: Cancers

Article Title: SHARPIN Enhances Ferroptosis in Synovial Sarcoma Cells via NF-κB- and PRMT5-Mediated PGC1α Reduction.

doi: 10.3390/cancers15133484

Figure Lengend Snippet: Figure 6. PRMT5 and NF-κB are essential regulators of ferroptosis downstream of SHARPIN. (A) Immunoblot and qPCR analyses of the effect of knockdown of SHARPIN on the expression levels of SDMA protein and IL-6 mRNA in Yamato cells. (B) The effect of a PRMT5 inhibitor, EPZ01566, on PGC1α, SLC7A11, and SDMA protein levels in Yamato cells. (C) The effect of a NF-κB inhibitor, SC- 514, on PGC1α protein, SLC7A11 protein, and IL-6 mRNA levels in Yamato cells. (D) Kaplan–Meier curve showing the relationship between DFS and SHARPIN and/or TFRC gene amplification (AMP), based on a TCGA dataset of all types of cancer. Subjects were divided into SHARPIN only AMP, TFRC only AMP, SHARPIN and TFRC AMP, and no AMP groups. (E) Kaplan–Meier curve showing the relationship between OS and SHARPIN and/or TFRC gene amplification (AMP), based on a TCGA dataset of soft tissue sarcoma samples. Subjects were divided into SHARPIN only AMP or HIGH (z-score ≥2), TFRC only AMP or HIGH, SHARPIN and TFRC AMP or HIGH, and no AMP or HIGH groups. (A,C) Statistical significance was calculated via a one-way ANOVA or Student’s t-test. Quantitative data are presented as the mean ± SD (n = 3). * p < 0.05. (D,E) Statistical significance was calculated using a log-rank test. The p-value is shown in each figure. The uncropped blots are shown in File S1.

Article Snippet: Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07- 412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology).

Techniques: Western Blot, Knockdown, Expressing

Journal: Nutrients

Article Title: Hydroxysafflor Yellow A Alleviates Acute Myocardial Ischemia/Reperfusion Injury in Mice by Inhibiting Ferroptosis via the Activation of the HIF-1α/SLC7A11/GPX4 Signaling Pathway

doi: 10.3390/nu15153411

Figure Lengend Snippet: Hydroxysafflor yellow A alleviated ferroptosis by activating the HIF-1α/SLC7A11/GPX4 signaling pathway. ( A ) The levels of ferroptosis-related proteins were assessed by Western blotting ( n = 3). ( B ) The total iron content in cardiac tissue was determined using a kit ( n = 6). ## p < 0.01 vs. the sham group; * p < 0.05, ** p < 0.01 vs. the MI/RI group.

Article Snippet: Anti-HIF-1α antibody (340462), anti-SLC7A11 antibody (382036), and goat antirabbit IgG secondary antibody (511203) were obtained from Chengdu Zen BioScience Co., Ltd. (Chengdu, China).

Techniques: Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Puerarin Ameliorates the Ferroptosis in Diabetic Liver Injure Through the JAK2/STAT3 Pathway Inhibition Based on Network Pharmacology and Experimental Validation

doi: 10.2147/DDDT.S487496

Figure Lengend Snippet: Effects of Puerarin on JAK2/STAT3 pathway and ferroptosis in mice with DLI. ( A-C ) p-JAK2 and p-STAT3 protein expression and the Western blot bands. ( D-J ) GPX4, ACSL4, PTGS2, SLC7A11, FTH1, and TFR1 protein expression and the Western blot bands. Data represent means ± SD (n = 5). Statistical analysis tested by one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control; ## P < 0.01, ### P < 0.001 vs model.

Article Snippet: Anti- NF-κB p65 (YT3108, 1:2000), anti-GPX4 (YN3047, 1:2000), anti-SLC7A11 (YT8130, 1:2000), anti-TFR1 (YT5374, 1:2000), anti-FTH1 (YT1692, 1:1000), and anti-ACSL4 (YT8070, 1:2000) were purchased from Immunoway (California, USA).

Techniques: Expressing, Western Blot, Control

Journal: Drug Design, Development and Therapy

Article Title: Puerarin Ameliorates the Ferroptosis in Diabetic Liver Injure Through the JAK2/STAT3 Pathway Inhibition Based on Network Pharmacology and Experimental Validation

doi: 10.2147/DDDT.S487496

Figure Lengend Snippet: Effects of Puerarin and AG490 on JAK2/STAT3 pathway and ferroptosis in HG-induced AML12 cells. ( A-C ) p-JAK2 and p-STAT3 protein expression and the Western blot bands. ( D-J ) GPX4, ACSL4, PTGS2, SLC7A11, FTH1, and TFR1 protein expression and the Western blot bands. Data represent means ± SD (n = 5). Statistical analysis tested by one-way ANOVA test. ** P < 0.01, *** P < 0.001 vs control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs model.

Article Snippet: Anti- NF-κB p65 (YT3108, 1:2000), anti-GPX4 (YN3047, 1:2000), anti-SLC7A11 (YT8130, 1:2000), anti-TFR1 (YT5374, 1:2000), anti-FTH1 (YT1692, 1:1000), and anti-ACSL4 (YT8070, 1:2000) were purchased from Immunoway (California, USA).

Techniques: Expressing, Western Blot, Control