Journal: Open Life Sciences

Article Title: TRIM21 accelerates ferroptosis in intervertebral disc degeneration by promoting SLC7A11 ubiquitination and degradation

doi: 10.1515/biol-2025-1216

Figure Lengend Snippet: TRIM21 decreases the expression of SLC7A11 by promoting the K48-linked ubiquitination of SLC7A11. (A) The interaction between TRIM21 and SLC7A11 proteins was analyzed by Co-IP. (B–E) NPCs were transfected with Flag-SLC7A11, HA-TRIM21, and WT/K48R/K63R His-UB. After IP with HA, immunoblotting was performed with anti-His. C–E: Quantification of SLC7A11-UB, HA-TRIM21, and Flag-SLC7A11 protein levels. (F–H) NPCs were transfected with His-UB, Flag-SLC7A11, and WT TRIM21 or MUT TRIM21. After IP with HA, immunoblotting was performed with anti-His. G–H: Quantification of SLC7A11-UB and Flag-SLC7A11 protein levels. (I–J) NPCs were transfected with TRIM21 overexpression plasmids or empty vectors, and treated with CHX for 0, 6, 12, 18, and 24 h. Protein levels of SLC7A11 were detected using immunoblotting. (K–L) NPCs were transfected with TRIM21 overexpression plasmids or empty vectors, treated with MG132, and then stimulated with CHX for 0, 6, 12, 18, and 24 h. Protein levels of SLC7A11 were detected using immunoblotting. All data are expressed as the means ± SD. ( n = 3 independent biological replicates/group in vitro experiments). Data were analyzed by one-way or two-way ANOVA with Tukey’s test for post hoc comparisons.

Article Snippet: To evaluate the protein stability of SLC7A11, transfected cells were pretreated with the proteasome inhibitor MG132 (20 μM, MedChemExpress) for 8 h, followed by exposure to cycloheximide (CHX, 100 μg/mL, MedChemExpress, USA) to inhibit de novo protein synthesis.

Techniques: Expressing, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Transfection, Western Blot, Over Expression, In Vitro

Journal: Open Life Sciences

Article Title: TRIM21 accelerates ferroptosis in intervertebral disc degeneration by promoting SLC7A11 ubiquitination and degradation

doi: 10.1515/biol-2025-1216

Figure Lengend Snippet: Knockdown of SLC7A11 reverses the inhibitory effects of TRIM21 knockdown on TBHP-induced ferroptosis in NPCs. (A) The mRNA level of SLC7A11 was detected by qPCR. Student’s t -test. (B) Cell viability was measured using the CCK-8 assay. (C–G) Levels of Fe 2+ , ROS, MDA, SOD, and GSH were detected using commercial assay kits. (H–K) The expression of ferroptosis-related proteins (GPX4, ACSL4, and TFRC) was analyzed by Western blot. I–K: Quantification of GPX4, ACSL4, and TFRC protein levels. All data are expressed as the means ± SD. ( n = 3 independent biological replicates/group in vitro experiments). Data were analyzed by one-way ANOVA with Tukey’s test for post hoc comparisons.

Article Snippet: To evaluate the protein stability of SLC7A11, transfected cells were pretreated with the proteasome inhibitor MG132 (20 μM, MedChemExpress) for 8 h, followed by exposure to cycloheximide (CHX, 100 μg/mL, MedChemExpress, USA) to inhibit de novo protein synthesis.

Techniques: Knockdown, CCK-8 Assay, Expressing, Western Blot, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Effect of Human Carbonic Anhydrase II on the Activity of the Human Electrogenic Na/HCO3 Cotransporter NBCe1-A in Xenopus Oocytes

doi: 10.1074/jbc.m602181200

Figure Lengend Snippet: FIGURE 6. Immunolocalization of EGFP and CA II domains in oocytes expressing EGFP-e1-CAII or EGFP- e1. Three days after injection of cRNA encoding EGFP-e1-CAII or EGFP-e1, oocytes were prepared for immu- nocytochemistryasindicatedunder“ExperimentalProcedures.”Preparationswereincubatedovernightsimul- taneously with mouse monoclonal anti-EGFP and with rabbit polyclonal anti-human CA II. The secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 594-conjugated goat anti-rab- bit. A-C, Alexa Fluor 488-labeled images were taken with excitation/emission filters set at band pass 488 nm/long pass 505 nm to detect EGFP signals. D-F, Alexa Fluor 594-labeled images were taken with excitation/ emission filters set at band pass 543 nm/long pass 560 nm to detect CA II signals. These images show that EGFP-e1-CAIIandEGFP-e1arecorrectlytargetedtotheplasmamembrane.G-I,mergesoftheimagesinthefirst and second columns. G shows that the EGFP staining (green) of EGFP-e1-CAII overlapped with the CA II staining (red). J–L, Nomarski images taken simultaneously with either the EGFP or CA II images. Bar 50 m.

Article Snippet: Slides were blocked by washing (once for 15 min) in TBS 0.1% BSA 10% normal goat serum (Vector Laboratories, Burlingame, CA).We incubated the preparations with mouse monoclonal anti-EGFP (BD Biosciences; 1:100 in TBS 0.1% BSA 10% goat serum) and rabbit polyclonal anti-human CA II (Rockland, Gilbertville, PA; 1:1000 in TBS 0.1% BSA 10% goat serum) overnight.

Techniques: Expressing, Injection, Labeling, Staining

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