Journal: JID Innovations

Article Title: Assessing Longitudinal Treatment Efficacies and Alterations in Molecular Markers Associated with Glutamatergic Signaling and Immune Checkpoint Inhibitors in a Spontaneous Melanoma Mouse Model

doi: 10.1016/j.xjidi.2024.100262

Figure Lengend Snippet: Quantifications and representative western immunoblots for the expression profiles of the molecular markers in melanomas. ( a ) mGluR1, ( b ) GLS, ( c ) xCT, ( d ) γ-H2AX, ( e ) EAAT2, ( f ) PD-L1, and ( g ) PD-1 bands were normalized to their respective tyrosinase bands, and these values were used for subsequent data analyses. The values in the table represent the average intensity of the protein band normalized to their respective tyrosinase band ± SEM. Below the tables are representative western blots associated with their respective proteins, where for mGluR1 and γ-H2AX, the immunoblots represent 12 weeks female mice, and for the GLS, xCT, EAAT2, PD-L1, and PD-1, the immunoblots represent 6 weeks female mice. The threshold used to define change/no change is associated with quantitative changes of 40–50% in protein expression observed between 6 and 18 weeks. At least 2 male and 2 female mice were used except for 6 weeks (male, anti–PD-1 and troriluzole + anti–PD-1; female, DMSO + rat IgG), 12 weeks (female, troriluzole and anti–PD-1), and 18 weeks xCT and EAAT2 (male, troriluzole + anti–PD-1) where only 1 mouse was used. These mice were randomly selected to be killed at 0, 6, 12, and 18 weeks to harvest livers and pigmented tumors. When pigmented tumors were harvested from each mouse, each specimen contained at least 4–6 pieces of independent tumors from that mouse, and western immunoblots were normalized to tyrosinase to consider only melanocytes/melanomas and not other cell types. To note, although we cannot completely exclude the contributions of melanocytes/nonmelanomas in the samples, we ensured during tumor harvest that only pigmented tumors were harvested with little to no adjacent normal skin present. GLS, glutaminase.

Article Snippet: For the westerns that used the antibodies, xCT (catalog number ANT-111, Alomone, Jerusalem, Israel), EAAT2 (catalog number A3679, Abclonal, Woburn, MA), tyrosinase (catalog number sc-20035, Santa Cruz Biotechnology), IRDye 680RD goat anti-rabbit IgG secondary antibody (catalog number 926-68071, LI-COR, Lincoln, NE), or IRDye 800CW goat anti-mouse IgG secondary antibody (catalog number 926-32210, LI-COR) were used.

Techniques: Western Blot, Expressing

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