Journal: MedComm

Article Title: ATR‐dependent ubiquitin‐specific protease 20 phosphorylation confers oxaliplatin and ferroptosis resistance

doi: 10.1002/mco2.463

Figure Lengend Snippet: USP20 de‐polyubiquitylates SLC7A11. (A) Huh‐7 cells transfected with the indicated shRNA were treated with MG132 for 6 h before collection. SLC7A11 was immunoprecipitated with anti‐SLC7A11 and immunoblotted with anti‐HA. (B) Immunoblotting was used to detect the ubiquitination of SLC7A11 in HEK293T cells cotransfected with Myc‐SLC7A11, HA‐ubiquitin and Flag‐USP20 (wild type or C154A). (C) USP20 removed the ubiquitin chain of SLC7A11 in a time‐ and dose‐dependent manner. (D) HA‐WT, K6, K11, K27, K29, K33, K48, or K63 Ub were cotransfected with Myc‐SLC7A11 and Flag‐USP20 into HEK293T cells. After treatment with 10 μM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of SLC7A11 detected by HA antibody. (E). Immunoblotting to detect the ubiquitination of the SLC7A11 deletion mutants (FL, ΔN, and ΔN) in HEK293T cells cotransfected with Myc‐SLC7A11 (FL, ΔN, and ΔN), HA‐Ub and USP20. (F) A schematic diagram of SLC7A11 and its mutants. (G) Immunoblotting to detect the ubiquitination of SLC7A11 mutants (K4, K12, K30, K37, K41, and K43) in HEK293T cells cotransfected with Myc‐SLC7A11 mutants, USP20 and HA‐Ub.

Article Snippet: Briefly, HA‐Ub, Myc‐SLC7A11, Flag‐USP20, or Flag‐USP20 C154A plasmid were transfected into HEK293T cells for 48 h and treated with 10 μM MG132 (MCE) for another 6 h. Cells were harvested at 4°C using RIPA extraction reagent (Meilun) after washed three times with prechilled PBS.

Techniques: Transfection, shRNA, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: Molecular Medicine Reports

Article Title: Reoxygenation induces reactive oxygen species production and ferroptosis in renal tubular epithelial cells by activating aryl hydrocarbon receptor

doi: 10.3892/mmr.2020.11679

Figure Lengend Snippet: AhR activation status does not affect Nrf2 activation or transcriptional activity. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative western blots of Nrf2 levels (corresponding to its activation status) and the expression of the Nrf2 transcriptional targets xCT (SLC7A11) and SOD-3. (B-D) Statistical analysis of the western blots. Neither Reox nor CH223191 affects Nrf2 activity, or the expression of xCT and SOD-3. Data are presented as the mean ± SEM of six independent experiments. AhR, arylhydrocarbon receptor; Nrf2, nuclear factor erythroid 2-related factor 2; xCT, cystine-glutamate antiporter; SOD-3, superoxide dismutase; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation.

Article Snippet: Primary antibodies were specific for AhR (1:200; cat. no. sc-133088; Santa Cruz Biotechnology, Inc.), cytochrome P450 family 1 subfamily A member 1 (CYP1A1; 1:500; cat. no. sc-25304; Santa Cruz Biotechnology, Inc.), Nrf2 (1:1,000; cat. no. TA343586; OriGene Technologies, Inc.), superoxide dismutase 3 (SOD-3; 1:100; cat. no. sc-271170; Santa Cruz Biotechnology, Inc.), cystine-glutamate antiporter (xCT, also known as SLC7A11; 1:1,000; cat. no. ANT-111; Alomone Labs), HIF-1α (1:500; cat. no. sc-10790; Santa Cruz Biotechnology, Inc.), LDH-A (1:1,000; cat. no. 2012; Cell Signaling Technology, Inc.), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Activity Assay, Cell Culture, Western Blot, Expressing, Analysis, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Bioinformatic analysis of SLC12A7 (A) Pan-cancer analysis results of SLC12A7 overexpression in multiple types of cancer (B,C) TCGA database indicated SLC12A7 overexpression in GC tissue samples and adjacent tissue samples (D) SLC12A7 mRNA expression was higher in GC tissues than in non-tumor tissues in GSE29272 , GSE3438 , GSE66229 datasets (E,F) ROC curve and OS analysis of SLC12A7 , indicating high SLC12A7 expression correlated with poor prognosis. ** represents p<0.01, *** represents p<0.001, ****represents p<0.0001.

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Analysis, Over Expression, Tissue, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Correlation between SLC12A7 mRNA expression and clinical pathological factors of GC patients in TCGA database.

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Overexpression of SLC12A7 in gastric cancer tissue samples compared to adjacent tissue samples (A) Immunohistochemistry detection of SLC12A7 staining for GC and adjacent tissues in GC tissue microarrays (B) SLC12A7 expression score chart in 77 pairs of GC and adjacent tissue samples (C) qRT-PCR indicated of mRNA expression level of SLC12A7 in GC tissues and corresponding adjacent tissues (D) Western blot was conducted on 47 pairs of GC and corresponding adjacent tissues to investigate the expression of SLC12A7 protein in GC tissues and adjacent tissues. All assays were performed in triplicate (** represents p < 0.01, P stands for adjacent tissues, T stands for GC tissues).

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Over Expression, Tissue, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Correlation between expression level of SLC12A7 and clinical pathological factors of GC patients in tissue microarrays.

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Expressing, Tissue

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Expression of SLC12A7 in gastric cancer cell lines before and after siRNA knockdown (A–C) mRNA expression levels and protein expression levels of SLC12A7 were elevated in all 2 GC cell lines HGC27, MGC803 compared with normal stomach epithelial cell line GES-1 (D) mRNA expression levels and protein expression levels of SLC12A7 in HGC27 cells (E) mRNA expression levels and protein expression levels of SLC12A7 in MGC803 cells. All assays were performed in triplicate (ns represents not significant; * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; **** represents p < 0.0001).

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Expressing, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Knockdown of SLC12A7 inhibited proliferation, metastasis, and EMT process in GC cell lines (A,B) Knockdown of SLC12A7 led to cancer proliferation inhibition in HGC27 and MGC803 cell lines (C,D) Knockdown of SLC12A7 reduced the migration and invasion of GC cells. (E,F) Knockdown of SLC12A7 exhibited prominent E-Cadherin upregulation and Vimentin downregulation. All assays were performed in triplicate (* represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001).

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Knockdown, Inhibition, Migration

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Knockdown of SLC12A7 led to cell cycle arrest (A,B) CDK1 and Cyclin A2 were downregulated in HGC27 and MGC803 cells after SLC12A7 inhibition (C,D) Flow cytometry results indicated an increase in the percentage of G2-phase cells and a decrease in the percentage of S-phase cells after SLC12A7 knockdown, suggesting G2/M cell-cycle arrest. All assays were performed in triplicate (* represents p < 0.05; ** represents p < 0.01).

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Knockdown, Inhibition, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Knockdown of SLC12A7 resulted in apoptosis in GC cells (A,C) Flow cytometry results showed elevated apoptosis rates in HGC27 and MGC803 cells after application of siRNA inhibition (B) Expression levels of apoptosis-related protein in HGC27 and MGC803 cells after SLC12A7 inhibition. All assays were performed in triplicate (ns represents not significant; * represents p < 0.05; *** represents p < 0.001; **** represents p < 0.0001).

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Knockdown, Flow Cytometry, Inhibition, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: Knockdown of SLC12A7 influenced PI3K/AKT signaling pathway in GC cells (A,B) Western blot results of PI3K and AKT total protein expression levels and phosphorylated protein expression levels in HGC27 and MGC803 cells after knocking down SLC12A7 . All assays were performed in triplicate (ns represents not significant; * represents p < 0.05; ** represents p < 0.01).

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Knockdown, Western Blot, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLC12A7 promotes malignant phenotypes of gastric cancer cells and is associated with Ezrin-related PI3K/AKT signaling

doi: 10.3389/fcell.2026.1828288

Figure Lengend Snippet: SLC12A7 knockdown attenuates PI3K/AKT phosphorylation and Ezrin expression in gastric cancer cells (A) Correlation analysis between SLC12A7 and Ezrin (B–D) Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, and Ezrin protein expression levels in HGC27 and MGC803 cells after co-transfected with si- SLC12A7 and Ezrin (E) Docking simulation of SLC12A7 and Ezrin molecules. All assays were performed in triplicate (ns represents not significant; * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001) (F) SLC12A7 and Ezrin show immunofluorescent colocalization in MGC803 (PCC = 0.80) and HGC27 (PCC = 0.69).

Article Snippet: For cell apoptosis rate detection, HGC27 and MGC803 cells were pretreated separately using si- SLC12A7 or si-NC for 48 h. A total of 1 × 10 6 cells at the logarithmic phase of growth were harvested according to manufacturer’s protocol: rinsed twice with PBS, each sample resuspended using a 100 μL 1×binding buffer, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) (MK1028, Boster, Wuhan, China) added and incubated on ice in the dark for 15 min. Another 400 μL of 1×binding buffer was added to resuspend the cells.

Techniques: Knockdown, Phospho-proteomics, Expressing, Analysis, Western Blot, Transfection, Docking Assay

Journal: bioRxiv

Article Title: Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

doi: 10.1101/2025.10.23.682519

Figure Lengend Snippet: A Schematic workflow of the high-throughput screening to identify ZIP14 inhibitors. B Chemical structure of 1-phenyl-8-(2-phenylethyl)-1,3,8-triazaspiro[4.5]decan-4-one (PPTD).

Article Snippet: The following primary antibodies were used: anti-human ZIP14 (generated previously ), anti-human ZIP8 (Alomone Labs, #AZT-008), and anti-β-actin (Cell Signaling Technology, #3700S).

Techniques: Sample Throughput, Screening

Journal: bioRxiv

Article Title: Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

doi: 10.1101/2025.10.23.682519

Figure Lengend Snippet: Xenopus oocytes injected either with ( A, B ) human ZIP14 or ( E, F ) human ZIP8 cRNA were incubated with either ( A, E ) ⁶⁵Zn or ( B, F ) ⁵⁵Fe in the presence of ( A, E ) 50 µM or ( B, F ) 10 µM PPTD for 30 min at 22 °C. Radioactivity was measured as described under Methods . Data are normalized against DMSO-treated transporter-expressing controls, and presented as mean ± standard deviation (S.D.) (n=3 independent experiments). Similarly, (C, D) TREx-hZIP14 or (G, H) TREx-hZIP8 cells were treated with Tet (1 μg/mL) for 24 h, followed by incubation either with ( C, G ) ⁵⁴MnCl₂ or (D, H) ¹⁰⁹CdCl₂ in the presence of PPTD at the indicated concentrations for 1 h. Radioactivity was measured as described under Methods . The data (mean + SD) represent results from three independent experiments. Statistical significance was determined using Student’s t -test (A, B, E, F) or one-way ANOVA followed by Dunnett’s post hoc test (C, D, G, H). (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ***** p < 0.0005).

Article Snippet: The following primary antibodies were used: anti-human ZIP14 (generated previously ), anti-human ZIP8 (Alomone Labs, #AZT-008), and anti-β-actin (Cell Signaling Technology, #3700S).

Techniques: Injection, Incubation, Radioactivity, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

doi: 10.1101/2025.10.23.682519

Figure Lengend Snippet: TREx-hZIP14 cells were treated with Tet (1 μg/mL) in combination with ( A ) ZnSO 4 (100 μM), ( B ) FeSO 4 (20 μM), ( C ) MnCl₂ (100 μM), or ( D ) CdCl₂ (20 μM), with or without PPTD at the indicated concentrations. Similarly, TREx-hZIP8 cells were treated under the same conditions ( E–H ). Phase-contrast images showing cell morphology ( A, B, E, F ). Cell viability ( C, D, G, H ) was quantified as described under Methods , and representative data (mean + SD) from three independent experiments are presented. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test ( C, D, G, H ). (* p < 0.05, *** p < 0.005, **** p < 0.001, ***** p < 0.0005). Scale bar: 20 μm.

Article Snippet: The following primary antibodies were used: anti-human ZIP14 (generated previously ), anti-human ZIP8 (Alomone Labs, #AZT-008), and anti-β-actin (Cell Signaling Technology, #3700S).

Techniques: Phase Contrast, Morphology