Journal: PLoS ONE

Article Title: SLC3A2 is a novel endoplasmic reticulum stress-related signaling protein that regulates the unfolded protein response and apoptosis

doi: 10.1371/journal.pone.0208993

Figure Lengend Snippet: SLC3A2 and GRP78 (A) mRNA and (B) expression as assessed by RT-qPCR and western blot analysis, respectively, in H9C2 and PC12 cells treated with DTT, TM, or TG for 4 h and in non-treated cells as controls. *** p < 0.001.

Article Snippet: For SLC3A2 and ATF4 immunostaining, cells were grown on glass coverslips, fixed with formalin, and treated with primary antibodies against SLC3A2 (Santa Cruz Biotechnology, sc-390154) and ATF4 (Abcam, ab194909) diluted in phosphate-buffered saline; 5% bovine serum albumin was added to block non-specific reactions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: SLC3A2 is a novel endoplasmic reticulum stress-related signaling protein that regulates the unfolded protein response and apoptosis

doi: 10.1371/journal.pone.0208993

Figure Lengend Snippet: Cells were immunostained with anti-SLC3A2 and anti-ATF4 antibodies, and nuclei were counterstained with DAPI. The fluorescence intensity was determined by confocal microscopy after 4 h of exposure to dimethyl sulfoxide (DMSO; control) or TG. Staining with or without SLC3A2 primary antibodies was performed as a positive or negative control.

Article Snippet: For SLC3A2 and ATF4 immunostaining, cells were grown on glass coverslips, fixed with formalin, and treated with primary antibodies against SLC3A2 (Santa Cruz Biotechnology, sc-390154) and ATF4 (Abcam, ab194909) diluted in phosphate-buffered saline; 5% bovine serum albumin was added to block non-specific reactions.

Techniques: Fluorescence, Confocal Microscopy, Control, Staining, Negative Control

Journal: PLoS ONE

Article Title: SLC3A2 is a novel endoplasmic reticulum stress-related signaling protein that regulates the unfolded protein response and apoptosis

doi: 10.1371/journal.pone.0208993

Figure Lengend Snippet: (A) Atf4 , Atf6 , Xbp1 , and Slc3a2 mRNA levels were measured by RT-qPCR in TG-treated and non-treated H9C2 cells pretreated with Slc3a2- siRNA or scrambled control. Target mRNA levels were normalized to β-actin expression. *** p < 0.001. (B) Slc3a2 mRNA levels were measured by RT-qPCR in TG-treated and non-treated H9C2 cells pretreated with siRNAs targeting Atf4 , Atf6 , or Xbp1 , or scrambled control. Slc3a2 mRNA expression was normalized to β-actin expression. ** p < 0.01, * p < 0.05.

Article Snippet: For SLC3A2 and ATF4 immunostaining, cells were grown on glass coverslips, fixed with formalin, and treated with primary antibodies against SLC3A2 (Santa Cruz Biotechnology, sc-390154) and ATF4 (Abcam, ab194909) diluted in phosphate-buffered saline; 5% bovine serum albumin was added to block non-specific reactions.

Techniques: Quantitative RT-PCR, Control, Expressing

Journal: PLoS ONE

Article Title: SLC3A2 is a novel endoplasmic reticulum stress-related signaling protein that regulates the unfolded protein response and apoptosis

doi: 10.1371/journal.pone.0208993

Figure Lengend Snippet: Atf4 , Atf6 , Xbp1 , and Slc3a2 mRNA levels were measured by RT-qPCR in TG-treated and non-treated H9C2 cells pretreated with rapamycin, Slc3a2-siRNA, or scrambled control siRNA. Target mRNA levels were normalized to β-actin expression. *** p < 0.001. mTOR1 mRNA levels were measured by RT-qPCR in TG-treated and non-treated H9C2 cells that were pretreated with siRNAs targeting Slc3a2 , Atf4 , Atf6 , or Xbp1 , or scrambled control. Target mRNA levels were normalized to β-actin expression. * p < 0.05.

Article Snippet: For SLC3A2 and ATF4 immunostaining, cells were grown on glass coverslips, fixed with formalin, and treated with primary antibodies against SLC3A2 (Santa Cruz Biotechnology, sc-390154) and ATF4 (Abcam, ab194909) diluted in phosphate-buffered saline; 5% bovine serum albumin was added to block non-specific reactions.

Techniques: Quantitative RT-PCR, Control, Expressing

Journal: PLoS ONE

Article Title: SLC3A2 is a novel endoplasmic reticulum stress-related signaling protein that regulates the unfolded protein response and apoptosis

doi: 10.1371/journal.pone.0208993

Figure Lengend Snippet: (A) Microscopic analysis of live cells following treatment with Slc3a2 siRNA or scrambled control. (B) Flow cytometry analysis of apoptosis in TG-treated and non-treated H9C2 cells pretreated with Slc3a2 siRNA or scrambled control. The cell population in the Q4 quadrant (Annexin V–/PI–) represents live cells, whereas the populations in the Q3 quadrant (Annexin V+/PI–) and the Q2 quadrant (Annexin V+/PI+) represent early- and late-apoptotic cells, respectively. The cell population in the Q1 quadrant (Annexin V–/PI+) represents necrotic cells. The sum of early- and late-apoptotic cells from three independent experiments is shown. *** p < 0.001 vs. control; ### p < 0.001 vs. TG treatment.

Article Snippet: For SLC3A2 and ATF4 immunostaining, cells were grown on glass coverslips, fixed with formalin, and treated with primary antibodies against SLC3A2 (Santa Cruz Biotechnology, sc-390154) and ATF4 (Abcam, ab194909) diluted in phosphate-buffered saline; 5% bovine serum albumin was added to block non-specific reactions.

Techniques: Control, Flow Cytometry

Journal: PLoS ONE

Article Title: SLC3A2 is a novel endoplasmic reticulum stress-related signaling protein that regulates the unfolded protein response and apoptosis

doi: 10.1371/journal.pone.0208993

Figure Lengend Snippet: (A) Heatmap of genes differentially expressed between control (left), scramble siRNA + TG-treated (middle), and Slc3a2 siRNA + TG-treated (right) cells. The data are log2-transformed. Statistical criteria for differential expression were a fold change ≥ 2 and a false discovery rate (FDR) < 0.05. (B) GO analysis using DAVID for the genes shown in (A). An FDR < 0.05 was considered statistically significant. (C) Ingenuity Pathway Analysis of the genes differentially expressed in the Slc3a2 siRNA-treated group revealing enrichment in signaling networks involved in protein processing in the ER. Genes most highly enriched in ER-related processes are indicated by a red star.

Article Snippet: For SLC3A2 and ATF4 immunostaining, cells were grown on glass coverslips, fixed with formalin, and treated with primary antibodies against SLC3A2 (Santa Cruz Biotechnology, sc-390154) and ATF4 (Abcam, ab194909) diluted in phosphate-buffered saline; 5% bovine serum albumin was added to block non-specific reactions.

Techniques: Control, Transformation Assay, Quantitative Proteomics

Journal: Cell death and differentiation

Article Title: Anaplastic Lymphoma Kinase signaling stabilizes SLC3A2 expression via MARCH11 to promote neuroblastoma cell growth.

doi: 10.1038/s41418-024-01319-0

Figure Lengend Snippet: Fig. 3 Knockdown of SLC3A2 suppresses NB cell growth and increases retinoic acid-induced neuronal differentiation. A–D CLB-BAR, CLB- GE, and CLB-GAR cells were treated with either control siRNA or SLC3A2-targeting siRNAs. A Cell growth was monitored by Incucyte every 6 h for 72-120 h (n = 3, mean ± s.e.m.). B Lysates collected at 72 h after siRNA transfection were immunoblotted with anti-SLC3A2, pAKT(S473), AKT, pERK (T202/Y204), ERK, PARP (asterisk (*) marked cleaved PARP), γH2A.X, α-tubulin and GAPDH. GAPDH was employed as immunoblotting loading control. Quantification of cleaved PARP (PARP*) and γH2A.X were performed with ImageJ (n = 3), shown in Supplementary Fig. 3. C Cell cycle analysis performed by NucleoCounter NC-3000 after 72 h (n = 3, mean ± s.e.m.). D Representative images showing differentiation morphology of CLB-GE cells 72 h post siRNA transfection. Scale bar = 50 µm. Bar graph indicates percentage of differentiated cells following siRNA treatment (n = 5, mean ± s.e.m). E, F SK-N-BE(2) cells treated with either SLC3A2-targeting or control siRNAs for 24 h, followed by retinoic acid (RA, 5 µM) for 48 h. E Representative images showing differentiation morphology of SK-N-BE(2) cells. Scale bar = 50 µm. Bar graph indicates percentage of differentiated cells (n = 6, mean ± s.e.m.). F Immunoblotting for neuronal differentiation markers (RET, DLG2) in SK-N-BE(2) cells (n = 3). GAPDH was employed as immunoblotting loading control. Immunoblot experiments were performed in biologically independent triplicates. Quantification was performed with ImageJ and analyzed by Student t test (unpaired, two- tailed). p values are indicated (****P < 0.0001, ***0.0001 < P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05, ns P ≥0.05); exact P-values are shown in Supplementary Fig. 3 and Supplementary Table 1.

Article Snippet: Primary antibodies against human SLC3A2 (#81977, was employed for immunoblotting, 1:2000; #13180, was used for immunoprecipitation), ALK (#3633, 1:2000), pALK (Y1278, #6941, 1:1000), ubiquitin (#3936, 1:1000), phospho-Tyrosine (p-Y-1000, #8954, 1:1000), pAKT (S473,#4060, 1:4000), AKT (#9272), pERK1/2 (Y204/T202, #4377, 1;2000), PARP (#9542, 1:1000), γH2A.X (Ser139, #9718, 1:1000), RET (#14556, 1:2000), DLG2 (#19046, 1:1000), SLC1A5 (ASCT2, #8057, 1:2000), SLC7A5 (LAT1, #32683, 1:1000), SLC7A11 (xCT, #12691, 1:1000), SLC38A1 (SNAT1, #36057, 1:2000), β-actin (#4970, 1:10000), GAPDH (#5174, 1:10,000) and α-tubulin (#2125, 1:10,000) were obtained from Cell Signaling Technology.

Techniques: Knockdown, Control, Transfection, Western Blot, Cell Cycle Assay, Two Tailed Test

Journal: Cell death and differentiation

Article Title: Anaplastic Lymphoma Kinase signaling stabilizes SLC3A2 expression via MARCH11 to promote neuroblastoma cell growth.

doi: 10.1038/s41418-024-01319-0

Figure Lengend Snippet: Fig. 5 Combined lorlatinib and AMXT-1501 treatment synergistically inhibits cell growth in ALK-driven NB and increases retinoic acid- induced neuronal differentiation. A IC50s of AMXT-1501 in CLB-BAR, CLB-GE, SK-N-BE(2) and SH-SY5Y cells after 5 d treatment (n = 3, mean ± s.e.m.). B CLB-BAR and CLB-GE cells were treated with lorlatinib and AMXT-1501 at the indicated concentrations and cell growth monitored by Incucyte every 6 h over a total of 120 h (n = 3, mean ± s.e.m.). The combination index value was calculated based on Chou- Talalay method using CompuSyn software [70]. C Immunoblot analysis of ALK signaling and apoptosis markers in CLB-GE cells treated with AMXT-1501 (0 or 8 µM) and lorlatinib (0 or 16 nM) as indicated for 24 h (n = 3). Lysates were immunoblotted with anti-ALK, pY1278-ALK, pAKT(S473), AKT, pERK (T202/Y204), ERK, PARP (asterisk (*) marked cleaved PARP), γH2A.X, α-tubulin and SLC3A2 and β-actin. β-actin was employed as immunoblotting loading control. Representative images of differentiation morphology of SK-N-BE(2) (D) and SH-SY5Y (E) cells after AMXT-1501 (0 or 8 µM) and RA (0 or 5 µM) treatment as indicated for 48 h or 24 h. Scale bar = 50 µm. Bar graph indicates percentage of differentiated cells (n = 3, mean ± s.e.m.). F Immunoblot analysis of neuronal differentiation markers (RET and DLG2) in SK-N-BE(2) and SH- SY5Y cells treated with AMXT-1501 (0 or 8 µM) and RA (0 or 5 µM) for 48 h or 24 h (n = 3). β-actin was employed as immunoblotting loading control. Immunoblot experiments (C and F) were performed in biologically independent triplicates. Quantifications were performed with ImageJ and analyzed by Student t-test (unpaired, two-tailed). p values are indicated (****P < 0.0001, ***0.0001 < P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05, ns P ≥0.05); exact P values are shown in Supplementary Fig. 4 and Supplementary Table 1.

Article Snippet: Primary antibodies against human SLC3A2 (#81977, was employed for immunoblotting, 1:2000; #13180, was used for immunoprecipitation), ALK (#3633, 1:2000), pALK (Y1278, #6941, 1:1000), ubiquitin (#3936, 1:1000), phospho-Tyrosine (p-Y-1000, #8954, 1:1000), pAKT (S473,#4060, 1:4000), AKT (#9272), pERK1/2 (Y204/T202, #4377, 1;2000), PARP (#9542, 1:1000), γH2A.X (Ser139, #9718, 1:1000), RET (#14556, 1:2000), DLG2 (#19046, 1:1000), SLC1A5 (ASCT2, #8057, 1:2000), SLC7A5 (LAT1, #32683, 1:1000), SLC7A11 (xCT, #12691, 1:1000), SLC38A1 (SNAT1, #36057, 1:2000), β-actin (#4970, 1:10000), GAPDH (#5174, 1:10,000) and α-tubulin (#2125, 1:10,000) were obtained from Cell Signaling Technology.

Techniques: Software, Western Blot, Control, Two Tailed Test

Journal: Cell death and differentiation

Article Title: Anaplastic Lymphoma Kinase signaling stabilizes SLC3A2 expression via MARCH11 to promote neuroblastoma cell growth.

doi: 10.1038/s41418-024-01319-0

Figure Lengend Snippet: Fig. 6 ALK signaling upregulates amino acid transporter systems. Gene oncology GSEA analysis of RNA-seq datasets. A NB tumors from either Alk-F1178S;Th-MYCN mice or Rosa26_Alkal2;Th-MYCN mice compared to Th-MYCN mice. B NB1 cells treated with either ALKAL2 or/and lorlatinib for 24 h. C CLB-BAR cells treated with lorlatinib for 24 h compared to DMSO treated control. D Heat map indicating biotinylation of SLC1A5, SLC3A2, SLC6A15, SLC38A1 in SK-N-AS.ALK-BirA* expressing cells compared to BirA* controls. Columns represent three biological replicates, data from [41]. Immunoblotting for anti-pALK (Y1278), SLC1A5, SLC3A2, SLC7A5, SLC7A11, SLC38A1 and GAPDH in NB1 cells treated with ALKAL2 (1 µg/ml) and lorlatinib (0 or 30 nM) for 24 h (E) and CLB-BAR cells treated with lorlatinib (0, 30 nM) for 24 h (F). G Schematic visualization of the regulation of SLC3A2 downstream of ALK in NB cells (created with BioRender.com). Immunoblot experiments were performed in biologically independent triplicates. Quantifications were performed with ImageJ and analyzed by Student t test (unpaired, two- tailed). p values are indicated (****P < 0.0001, ***0.0001 < P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05, ns P ≥0.05); exact P values are shown in Supplementary Fig. 5 and Supplementary Table 1.

Article Snippet: Primary antibodies against human SLC3A2 (#81977, was employed for immunoblotting, 1:2000; #13180, was used for immunoprecipitation), ALK (#3633, 1:2000), pALK (Y1278, #6941, 1:1000), ubiquitin (#3936, 1:1000), phospho-Tyrosine (p-Y-1000, #8954, 1:1000), pAKT (S473,#4060, 1:4000), AKT (#9272), pERK1/2 (Y204/T202, #4377, 1;2000), PARP (#9542, 1:1000), γH2A.X (Ser139, #9718, 1:1000), RET (#14556, 1:2000), DLG2 (#19046, 1:1000), SLC1A5 (ASCT2, #8057, 1:2000), SLC7A5 (LAT1, #32683, 1:1000), SLC7A11 (xCT, #12691, 1:1000), SLC38A1 (SNAT1, #36057, 1:2000), β-actin (#4970, 1:10000), GAPDH (#5174, 1:10,000) and α-tubulin (#2125, 1:10,000) were obtained from Cell Signaling Technology.

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Two Tailed Test

Journal: Antiviral research

Article Title: Harnessing host enhancers of SARS-CoV-2 entry as novel targets for antiviral therapy.

doi: 10.1016/j.antiviral.2024.105951

Figure Lengend Snippet: Fig. 4. SARS-CoV-2 regulates the expression of some of the target proteins. A) Cell lysates were separated by SDS-PAGE and analyzed by Western blot depicting the expression of target proteins and SARS-CoV-2 N, 24 h post infection with pre-VOC B.1 and Omicron BA.1 at MOI 1. A representative blot of three independent experiments is shown. Equal loading was confirmed by probing for beta actin. B-G) Quantification of three independent infection experiments as shown in (A) for ACE2, ATP1B1, SLC3A2, ADAM10, EGFR, PLNXB2 across B to G, respectively. Statistical significance was determined with a one-way ANOVA test.

Article Snippet: Rabbit polyclonal against ITGB4 (#21738-1- AP), mouse monoclonal against ALCAM (#67768-1-Ig), rabbit polyclonal against PLXNB2 (#10602-1-AP), rabbit polyclonal against SLC3A2 (#15193-1-A), mouse monoclonal EGFR (#66455-1-Ig), mouse monoclonal EPHA2 (#66736-1-Ig), rabbit polyclonal ITGAV (#27096- 1-AP) and rabbit polyclonal anti-goat IgG HRP antibody (#SA00001-4) were purchased from Proteintech.

Techniques: Expressing, SDS Page, Western Blot, Infection

Journal: Frontiers in Oncology

Article Title: Decrease of Intracellular Glutamine by STF-62247 Results in the Accumulation of Lipid Droplets in von Hippel-Lindau Deficient Cells

doi: 10.3389/fonc.2022.841054

Figure Lengend Snippet: Effects of STF-62247 on glutamine transporters. (A, B) mRNA relative expression, measured by RT-qPCR, of glutamine transporters SLC1A5, SLC7A5 and SLC3A2 influenced by (A) VHL status and (B) STF-62247. RCC4 VHL- and VHL+ were treated with 1.25 µM STF-62447 for 24 hr. Results for treated cells are compared to control cells (represented by the dotted line at 1). Results are presented as means and SEM of three independent experiments. (C) Glutamine transporters, SLC1A5, SL7A5 and SLC3A2, protein levels in RCC4 VHL- and VHL+ treated to STF-62247 for 24 and 48 hr. (D) Intracellular leucine was measured by LC-MS in RCC4 VHL- and VHL+ treated with 1.25 µM STF-62247 for 24 hr (N=6). Student’s t -tests were performed to compare results between VHL- and VHL+ cells (A) or between controls and treated cells (B, D) (*p < 0.05, **p < 0.01, ***p < 0.001). (E) Cell viability was evaluated by XTT assay in RCC4 VHL- and VHL+ cells. SLC1A5 and SLC7A5 inhibitors, GPNA and BCH respectively, were tested alone (concentrations from 0 to 1 mM) or combined with 1.25 µM STF-62247 (N=3). Two-Way ANOVA followed by Tukey’s test was performed to assess statistically significant results. Comparison between CTL and STF-62247 conditions for each concentration of inhibitor (GPNA or BCH) are denoted with the following statistical marks *p < 0.05, **p < 0.01 or ***p < 0.001. Comparison of each inhibitor concentrations (x-axis) are made with their respective control (0 mM columns, with or without STF-62247) and statistical significances are denoted by # p < 0.05, ## p < 0.01 or ### p < 0.001.

Article Snippet: Membranes were blocked using 5% skim milk diluted in a solution of 0.075% PBS-Tween (PBS-T) and incubated overnight in 3% BSA with specific primary antibodies against VHL, HIF-1α and HIF-2α (Cell signaling #68547, 14179, 59973), SLC1A5 and β-actin (Santa Cruz Biotechnologies sc-99002 and sc-47778), SLC7A5 (Medical & Biological Laboratories #BMP011), SLC3A2 (Aviva Systems Biology #OAAB00158), ASNS (Signalway #32909), SCD1 (Applied Biological Materials #ABM-G076), PLIN2 (Proteintech #15294-1-AP) and CPT1A (Abcam #ab220789).

Techniques: Expressing, Quantitative RT-PCR, Control, Liquid Chromatography with Mass Spectroscopy, XTT Assay, Comparison, Concentration Assay

Journal: bioRxiv

Article Title: Newcastle disease virus induced ferroptosis through p53-SLC7A11-GPX4 axis mediated nutrient deprivation in tumor cells

doi: 10.1101/2021.01.03.424919

Figure Lengend Snippet: (A) Cell lysates were analyzed for the levels of p53, SLC7A11, SLC3A2, GPX4, FTH1 and NP, in an MOI dependent manner by western blot. β-Actin was used as the loading control. Erastin (5 μM) was added as a ferroptosis inducer. (B) Western blot analyses of the levels of p53, SLC7A11, SLC3A2, GPX4, NP, and FTH1 in U251 cells, with timepoints indicated. β-actin was served as a loading control. (C) mRNA levels of SLC7A1, p53, FTH1, and GPX4 in U251 cells treated with NDV (MOI = 1). (D) Detection of intracellular GSH concentration after NDV infection. (E) Cell lysates were analyzed for the levels of ALOX12 and NP in a time-dependent manner with western blotting. β-Actin was included as the loading control. All experiments were performed at least three times. Significance was analyzed using a two-tailed Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies against FTH1 (#4393), TFR1 (#13208), and SLC3A2 (#47213), SignalSilence® Control siRNA (Fluorescein Conjugate) (#6201), and SignalSilence® p53 siRNA I (#6231) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Control, Concentration Assay, Infection, Two Tailed Test

Journal: Apoptosis : an international journal on programmed cell death

Article Title: EFNA4-enhanced deubiquitination of SLC7A11 inhibits ferroptosis in hepatocellular carcinoma.

doi: 10.1007/s10495-024-02042-4

Figure Lengend Snippet: Fig. 3 EFNA4 promotes HCC proliferation and metastasis by inhibiting ferroptosis in HCC cells with EPHA2 receptor knocked out. A–B Transcrip- tome RNA sequencing: volcano plot and heatmap showing dif- ferentially expressed genes. C KO enrichment analysis; D GO analysis; E Reactome enrich- ment analysis of differentially expressed genes. F The top 10 matching proteins according to the mass spectrometry results. G Association of SLC7A11 with SLC3A2 in HCC patients. H TNM stage and OS. I Cor- relation analysis of EFNA4, SLC3A2 and SLC7A11; *p < 0.05, ***p < 0.001. J 10 µM erastin was added for 0-, 5- or 10-h cell treatment, fol- lowed by staining using 7-ami- noactinomycin (7-AAD) and annexin V for flow cytometry experiments to detect ferroptosis levels. (K) Electron microscopic examination revealed mitochon- drial ultrastructure in different genotype variants of HCC-LM3 cells. Quantitative analysis was performed to determine the number of cristae within each mitochondrion. The mitochon- drial cristae (highlighted by red arrows) were clearly visible in the images. Scale bars, 2 μm and 500 nm. L ROS levels in the EFNA4-overexpressing and Crsiper/Cas9 EPHA2-over- expressing HCC models were detected by flow cytometry and the statistical analysis. M Western blot analysis of GPX4 levels in cells overexpressing EFNA4 and in the Crsiper/ Cas9 EPHA2 HCC model. N–O MDA and intracellular ferrous ions assays. P Western blotting was used to assess the EFNA4- USP9X-SLC7A11 axis in vivo experiments (n = 5)

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH;60,004-1-ig), N-cadherin (22,018-1-AP), vimentin (10,366-1-AP), EPHA2 (66,736-1-Ig), SLC7A11 (26,864-1-AP; R), SLC7A11 (68,785-1-PBS; M), GPX4 (67,763-1-ig), Ki67 (27,309-1-AP), SLC3A2 (15,193-1- AP; R), SLC3A2 (66,883-1-ig; M), GST (HRP-66001) and His (66,005-1-ig) were provided by Proteintech (Wuhan, China).

Techniques: RNA Sequencing, Mass Spectrometry, Staining, Flow Cytometry, Expressing, Western Blot, In Vivo

Journal: Apoptosis

Article Title: EFNA4-enhanced deubiquitination of SLC7A11 inhibits ferroptosis in hepatocellular carcinoma

doi: 10.1007/s10495-024-02042-4

Figure Lengend Snippet: EFNA4 promotes HCC proliferation and metastasis by inhibiting ferroptosis in HCC cells with EPHA2 receptor knocked out. A – B Transcriptome RNA sequencing: volcano plot and heatmap showing differentially expressed genes. C KO enrichment analysis; D GO analysis; E Reactome enrichment analysis of differentially expressed genes. F The top 10 matching proteins according to the mass spectrometry results. G Association of SLC7A11 with SLC3A2 in HCC patients. H TNM stage and OS. I Correlation analysis of EFNA4, SLC3A2 and SLC7A11; * p < 0.05, *** p < 0.001. J 10 µM erastin was added for 0-, 5- or 10-h cell treatment, followed by staining using 7-aminoactinomycin (7-AAD) and annexin V for flow cytometry experiments to detect ferroptosis levels. (K) Electron microscopic examination revealed mitochondrial ultrastructure in different genotype variants of HCC-LM3 cells. Quantitative analysis was performed to determine the number of cristae within each mitochondrion. The mitochondrial cristae (highlighted by red arrows) were clearly visible in the images. Scale bars, 2 μm and 500 nm. L ROS levels in the EFNA4-overexpressing and Crsiper/Cas9 EPHA2-overexpressing HCC models were detected by flow cytometry and the statistical analysis. M Western blot analysis of GPX4 levels in cells overexpressing EFNA4 and in the Crsiper/Cas9 EPHA2 HCC model. N – O MDA and intracellular ferrous ions assays. P Western blotting was used to assess the EFNA4-USP9X-SLC7A11 axis in vivo experiments ( n = 5)

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH;60,004-1-ig), N-cadherin (22,018-1-AP), vimentin (10,366-1-AP), EPHA2 (66,736-1-Ig), SLC7A11 (26,864-1-AP; R), SLC7A11 (68,785-1-PBS; M), GPX4 (67,763-1-ig), Ki67 (27,309-1-AP), SLC3A2 (15,193-1-AP; R), SLC3A2 (66,883-1-ig; M), GST (HRP-66001) and His (66,005-1-ig) were provided by Proteintech (Wuhan, China).

Techniques: RNA Sequencing, Mass Spectrometry, Staining, Flow Cytometry, Western Blot, In Vivo

Journal: Apoptosis

Article Title: EFNA4-enhanced deubiquitination of SLC7A11 inhibits ferroptosis in hepatocellular carcinoma

doi: 10.1007/s10495-024-02042-4

Figure Lengend Snippet: EFNA4 interacts with SLC7A11 to enhance HCC cell growth and metastasis with EPHA2 receptor knocked out. A Co-IP was conducted for detecting interaction of EFNA4, SLC3A2 and SLC7A11. B GST-EFNA4 interacts with His-SLC7A11 in vitro according to a GST pull-down assay. C IF was used to detect the interaction of EFNA4, SLC3A2 and SLC7A11 (1000 ×, scale bar = 10 μm). D Co-IP assay showing an interaction between EFNA4 (GST-EFNA4-161-201) and WT-SLC7A11 in HCC cell lines and an interaction between SLC7A11 (His-SLC7A11-222-501) and WT-EFNA4 in HCC cell lines

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH;60,004-1-ig), N-cadherin (22,018-1-AP), vimentin (10,366-1-AP), EPHA2 (66,736-1-Ig), SLC7A11 (26,864-1-AP; R), SLC7A11 (68,785-1-PBS; M), GPX4 (67,763-1-ig), Ki67 (27,309-1-AP), SLC3A2 (15,193-1-AP; R), SLC3A2 (66,883-1-ig; M), GST (HRP-66001) and His (66,005-1-ig) were provided by Proteintech (Wuhan, China).

Techniques: Co-Immunoprecipitation Assay, In Vitro, Pull Down Assay

Journal: Apoptosis

Article Title: EFNA4-enhanced deubiquitination of SLC7A11 inhibits ferroptosis in hepatocellular carcinoma

doi: 10.1007/s10495-024-02042-4

Figure Lengend Snippet: EFNA4 recruits the deubiquitinase USP9X to regulate SLC7A11 protein stability with EPHA2 receptor knocked out. A SLC7A11 protein expression was measured through Western blot in EPHA2 knockout and EFNA4-overexpressing HCC cell lines. B – C Effect of MG132 and CHX treatment for varying durations on SLC7A11 protein stability. D Western blotting was used to assess the impact of SLC7A11 deubiquitination. E Co-IP was carried out for detecting interaction of EFNA4, SLC7A11 and USP9X. F qRT‒PCR was performed for testing the USP9X knockdown efficiency. G Western blotting was used to assess SLC7A11 deubiquitination levels in HCC cell lines with USP9X knockdown and EFNA4 overexpression

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH;60,004-1-ig), N-cadherin (22,018-1-AP), vimentin (10,366-1-AP), EPHA2 (66,736-1-Ig), SLC7A11 (26,864-1-AP; R), SLC7A11 (68,785-1-PBS; M), GPX4 (67,763-1-ig), Ki67 (27,309-1-AP), SLC3A2 (15,193-1-AP; R), SLC3A2 (66,883-1-ig; M), GST (HRP-66001) and His (66,005-1-ig) were provided by Proteintech (Wuhan, China).

Techniques: Expressing, Western Blot, Knock-Out, Co-Immunoprecipitation Assay, Knockdown, Over Expression

Journal: Apoptosis

Article Title: EFNA4-enhanced deubiquitination of SLC7A11 inhibits ferroptosis in hepatocellular carcinoma

doi: 10.1007/s10495-024-02042-4

Figure Lengend Snippet: Ferroptosis increased in HCC cells with USP9X knockdown. A 10 µM erastin was added to treat cells for 0, 5 or 10 h, followed by staining using 7-aminoactinomycin (7-AAD) and annexin V for flow cytometry to detect ferroptosis levels; * p < 0.05. B – C ROS and Western blot analyses were carried out for detecting ferroptosis-related ROS and GPX4 levels in cells with overexpression of EFNA4 or knockdown of USP9X in an HCC model. D – E MDA and intracellular ferrous ions assays. F IHC was used to detect ferroptosis-related GPX4 and SLC7A11 in HCC models overexpressing EFNA4 and expressing different USP9X. G HE staining and IHC were used to detect the growth and metastasis of HCC cells overexpressing EFNA4 and expressing different USP9X. H Western blotting was used to assess the EFNA4-USP9X-SLC7A11 axis in vivo experiments ( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH;60,004-1-ig), N-cadherin (22,018-1-AP), vimentin (10,366-1-AP), EPHA2 (66,736-1-Ig), SLC7A11 (26,864-1-AP; R), SLC7A11 (68,785-1-PBS; M), GPX4 (67,763-1-ig), Ki67 (27,309-1-AP), SLC3A2 (15,193-1-AP; R), SLC3A2 (66,883-1-ig; M), GST (HRP-66001) and His (66,005-1-ig) were provided by Proteintech (Wuhan, China).

Techniques: Knockdown, Staining, Flow Cytometry, Western Blot, Over Expression, Expressing, In Vivo

Journal: Apoptosis

Article Title: EFNA4-enhanced deubiquitination of SLC7A11 inhibits ferroptosis in hepatocellular carcinoma

doi: 10.1007/s10495-024-02042-4

Figure Lengend Snippet: Schematic diagram showing the mechanism of EFNA4 with SLC7A11/SLC3A2/USP9X ferroptosis pathway. EFNA4 binds to SLC7A11, a well-known oncogene, which has been widely reported to inhibit ferroptosis through its structural domain (a.a. 161–201), and further promotes deubiquitination and inhibits degradation of SLC7A11 by recruiting the deubiquitinase USP9X

Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH;60,004-1-ig), N-cadherin (22,018-1-AP), vimentin (10,366-1-AP), EPHA2 (66,736-1-Ig), SLC7A11 (26,864-1-AP; R), SLC7A11 (68,785-1-PBS; M), GPX4 (67,763-1-ig), Ki67 (27,309-1-AP), SLC3A2 (15,193-1-AP; R), SLC3A2 (66,883-1-ig; M), GST (HRP-66001) and His (66,005-1-ig) were provided by Proteintech (Wuhan, China).

Techniques: