Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) SH3 domain GRB2-like endophilin B1 (SH3GLB1) and superoxide dismutase 2 (SOD2) levels were simultaneously enhanced in the patient derived xenografts (PDX) model of naïve glioblastoma (GBM) tumors after temozolomide (TMZ) (5 mg/kg) treatment for three weeks. ( B ) The results show increased levels of 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining (a reactive oxygen species (ROS) detection probe) in parental U87MG (for 6 h) or A172 (for 24 h) cells after triple co-incubation with TMZ, ATZ, and HNE. H 2 O 2 : 100 μM. Scale bar: 50 μm

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Derivative Assay, Staining, Incubation

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: SH3 domain GRB2-like endophilin B1 (SH3GLB1) is a downstream protein of superoxide dismutase 2 (SOD2). ( A ) The biological network of SH3GLB1-mediated autophagy and SOD2-involved antioxidants was predicted using the STRING bioinformatics tool. ( B ) Resistant cells were transfected with the microtubule-associated protein 1 light chain 3-Enhanced Green Fluorescent Protein (LC3-EGFP) plasmid, and representative fluorescent images for the formation of LC3-EGFP dots (puncta) are shown. Scale bar: 4 μm. ( C ) Protein immunoblotting showing that the resistant cells treated with temozolomide (TMZ) for 24 h induced autophagic reactions, which were attenuated by pretreatment with diethyldithiocarbamic acid (DETC; an SOD inhibitor). ( D ) Immunohistochemical (IHC) staining demonstrating SH3GLB1 expression in primary, recurrent glioblastoma (GBM-R) cells inoculated subcutaneously into mice receiving the indicated treatments for 15 days. A statistical graph is shown in the right-hand panel. Scale bar: 200 μm. ( E ) SOD2 siRNA or ( F ) overexpression vectors were used in U87MG- and A172-resistance cells or U87MG- and A172-parental cells, respectively, three days after transfection to examine the association between SOD2 and SH3GLB1. ( G ) SH3GLB1 siRNA was used in U87MG- and A172-resistance cells, and the association between SH3GLB1 and SOD2 was studied using western blotting. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunohistochemical staining, Immunohistochemistry, Expressing, Over Expression, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: SH3GLB1 levels are regulated by hydrogen peroxide. ( A ) Blots show that co-treatment with TMZ + 4-hydroxynonenal (HNE) + 3-amino-1,2,4-triazole (ATZ), enhanced the expression of SH3GLB1 in parental cells. ( B ) Cells were pretreated with N-acetyl-L-cysteine (NAC) and subjected to three co-treatments. U87MG cells were co-treated for 8 h and A172 cells for 18 h. Intracellular H 2 O 2 levels were measured, and ( C ) SH3GLB1, p-AKT (Ser473), and SOD2 levels were detected by western blotting. ( D ) MK-2206 inhibits p-Akt (Ser473) expression. ( E ) Blots showing the levels of the indicated proteins after co-treatment with TMZ, SC-79 (2-Amino-6-chloro-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester), and NAC. TMZ: 100 μM, ATZ: 20 mM, HNE: 10 μM, NAC: 10 mM, MK-2206: 5 μ, SC-79: 10 μg/mL. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: Increased levels of hydrogen peroxide demonstrate different effects on SH3GLB1 expression in the resistant cells. ( A ) MK-2206 was pretreated in the TMZ-treated resistant cells. ( B ) IHC staining demonstrating p-AKT levels in U87MG-R cells transfected with shSH3GLB1 or shControl vectors and inoculated subcutaneously into mice receiving the indicated treatments for 23 days. A statistical graph is shown in the right-hand panel. The arrows indicate the positive staining of SH3GLB1. Scale bar: 200 μm. ( C ) The resistant cells were treated with the indicated reagents. U87MG-R cells were co-treated for 18 h and A172-R cells for 8 h. The levels of intracellular H 2 O 2 were measured. ( D ) Protein immunoblotting after stimulation and rescue treatments. ( E ) Resistant cells were co-treated with TMZ and NAC. TMZ: 100 μM, MK-2206: 5 μM, ATZ: 20 mM, HNE: 10 μM, NAC: 10 mM, MK-2206: 5 μM. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Immunohistochemistry, Transfection, Staining, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) Blots showing the levels of the indicated proteins. Resistant cells were treated with TMZ with or without MK-2206. TMZ: 100 μM, MK-2206: 5 μM ( B ) The parental and resistant cells are treated with increasing concentrations of extracellular H 2 O 2 for 24 h. H 2 O 2 concentrations are indicated. The summary graph demonstrates the difference in the expression of SH3GLB1 in response to extracellular H 2 O 2 between the two cell lines. ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: TMZ combined with the inhibitors of an H 2 O 2 -related enzyme affects autophagy levels and tumor growth in the resistant cells. ( A ) The triple co-treatment caused simultaneous changes in autophagy and SH3GLB1 expression. ( B ) Proliferation assay results for parental or resistant cells treated with the indicated compounds are shown as bar graphs, suggesting that the resistant cells were more susceptible to H 2 O 2 accumulation. ( C ) Morphology of A172 and A172-R cells after 72 h of treatment. Control group is no TMZ-treated group. Scale bar: 100 μm. ( D ) Arrows indicate the inhibition of SH3GLB1 levels in the TMZ+HNE and TMZ+HNE+ATZ groups. ( E ) The mice were subcutaneously injected with luciferase-expressing U87MG-R cells. Bioluminescence signals were recorded on the indicated days using an IVIS imaging system. The growth curves of the tumors were analyzed according to the bioluminescence intensity. (N = 5 in each group) ( F ) Immunoblots showing the protein levels of xenograft tumor lysates from mice harvested after the indicated treatments. TMZ: 5 mg/kg, HNE: 2.5 mg/kg ( G ) The IHC staining demonstrates autophagy levels in shSH3GLB1 or shControl group of U87MG-R cells subcutaneously injected in mice and those receiving the indicated treatments. The statistic graph is shown in the right panel. Scale bar: 200 μm. Scale bar in the enlarged graph represents 1 mm. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3~5 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Proliferation Assay, Control, Inhibition, Injection, Luciferase, Imaging, Western Blot, Immunohistochemistry

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: Mitochondrial dysfunction affects SH3GLB1 expression via H 2 O 2 /AKT signaling. ( A ) H 2 O 2 levels in resistant cells were measured after the indicated treatments. TMZ: 100 μM, CCCP: 10 μM ( B ) The protein immunoblot shows that SH3GLB1 levels are regulated by treating with CCCP for 24 h with or without TMZ. Resistant cells (U87MG-R) were treated with TMZ with or without HgCl 2 . ( C ) The statistic graph shows H 2 O 2 levels in the indicated treatments. ( D ) The blots demonstrate the indicated protein expression after the treatments. The statistic graphs are shown. TMZ: 100 μM, HgCl 2 : 20 μM. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) SH3GLB1 and Bax levels were compared between normal and GBM tissues from the TCGA-GBM dataset. ( B ) The protein immunoblots showed levels of Bax-α (21 kd; the lower arrow) and Bax-β (24 kd; the upper arrow) in the parental cells and the derived resistant cells. Corresponding fold-change values (relative to control) are shown in the lower box. ( C ) The western blotting showed that the resistant cells (A172-R) were treated with TMZ with or without HgCl 2 . TMZ: 100 μM, HgCl 2 : 20 μM. Corresponding fold-change values (relative to control) are shown beneath the Western blot panels. N = 3 in each group

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Western Blot, Derivative Assay, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: TMZ elevates reactive oxygen species (ROS) levels, including H 2 O 2 . In resistant GBM cells, moderate H 2 O 2 activates AKT, driving SH3GLB1 expression and sustaining drug resistance, whereas excessive H 2 O 2 (e.g., following HNE co-treatment) suppresses SH3GLB1 and impairs resistance. Furthermore, HgCl 2 downregulates mitochondrial aquaporin-9 (AQP9), reducing H 2 O 2 flux and SH3GLB1 expression. This schematic illustrates how H 2 O 2 levels, AKT activation, and AQP9 modulation collectively shape SH3GLB1-driven resistance

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ENDOU-1-induced cytoplasmic HnRNPA3 recognizes m6A methylation on the upstream reading frame of human CHOP transcripts to achieve maximal CHOP translation

doi: 10.1007/s00018-026-06180-7

Figure Lengend Snippet: Screening and identification of putative proteins that interact with zebrafish Endouc, human ENDOU-1 and human CHOP uORF RNA. A The pull-down assay. Total lysates extracted from HEK293T cells were pulled down by recombinant zebrafish Endouc fused with Flag (Ec-Flag) produced from Sf21 insect cells. The precipitated proteins were analyzed on SDS-PAGE by silver staining: lane m, protein markers; lane ctrl, protein profiles of mixture containing Flag beads, extracts of Sf21 wild-type (Wt) cells and total lysates extracted from HEK293T cells served as control; and lane Ec-Flag, protein profiles of mixture containing Flag beads, Ec-Flag produced by Sf21 and total lysates extracted from HEK293T cells. Brackets on the right represented the gel slices excised for LC-MS/MS. B Four putative proteins that interacted with zebrafish Endouc-Flag (Ec-Flag) were screened by co-immunoprecipitation (Co-IP). HEK293T cells were transfected with empty pCS2 vector or pCS2 vector to express Flag-tagged zebrafish Endouc (Ec-Flag), or Myc-tagged human HnRNPA3 (HnRNPA3-Myc), human Sec61α1 (Sec61α1-Myc), human Srsf9 (Srsf9-Myc), and human SREK1IP1 (SREK1IP1-Myc). Cell lysates were subjected to IP with anti-Flag-HRP antibody or anti-Myc-HRP antibody. The presence of Flag- and Myc-tagged recombinant proteins in cell extracts prior to IP was controlled using anti-Flag and anti-Myc antibodies (Input). Co-IP of ( C ) zebrafish Endouc (Ec-Flag) or ( D ) human ENDOU-1 (ENDOU-1-Flag) with human HnRNPA3 (HnRNPA3-Myc). Either Myc-tagged human HnRNPA3 (HnRNPA3-Myc), Flag-tagged zebrafish Endouc (Ec-Flag) or human ENDOU-1 (ENDOU-1-Flag) produced by HEK293T cells was subjected to IP. E Electrophoretic mobility shifted assay of RNA. Biotin-labeled h uORF chop transcript was reacted with an increased amount of HnRNPA3-Myc. The positions of free RNA (free probe) and RNA–protein complex (shifted band) were indicated on the right. The luc 105‐nt RNA and single‐stranded h uORF chop 105‐nt DNA served as negative controls. F Western blot analysis after RNA pull-down assay to investigate the interaction between HnRNPA3 and biotin-labeled RNA, as indicated. HnRNPA3-Flag-expressing HEK293T cells were used to collect the protein lysate for RNA pulldown and analysis by immunoblotting with Flag antibody. HnRNPA3 exhibited more affinity to h uORF chop RNA than that of either uORF atf4 RNA or luc RNA

Article Snippet: Antibodies were CoraLite ® Plus 488-conjugated HNRNPA3 Polyclonal antibody (Proteintech, CL488-25142; 1:200) and anti-PP11 antibody (Abcam, ab18520; 1:100) conjugated with Alexa Fluor ® 647 using Alexa Fluor ® 647 Conjugation kit (Abcam, ab269823).

Techniques: Pull Down Assay, Recombinant, Produced, SDS Page, Silver Staining, Control, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Labeling, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ENDOU-1-induced cytoplasmic HnRNPA3 recognizes m6A methylation on the upstream reading frame of human CHOP transcripts to achieve maximal CHOP translation

doi: 10.1007/s00018-026-06180-7

Figure Lengend Snippet: HnRNPA3 is positively correlated with CHOP translation. A Schematic showing the dual-luc reporter constructs for measuring huORF chop -mediated translational inhibition. phRG-TK was used as an internal control. B Dual-luc assay was used to analyze the effect of HnRNPA3 on h uORF chop -MTI. Histograms present the luc activity obtained from HEK293T cells co-transfected with puORFchop‐luc, phRG‐TK, and each indicated plasmid and then treated with either DMSO (control group; grey column) or Thapsigargin (TH; stress group; solid column) for 6 h, followed by analysis of luc activity. Cells transfected with pCS2vector and kept at normal conditions served as a control group. Relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from the control group normalized to 1. Data were averaged from three independent trials and presented as mean ± SEM. ***P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test.) ( C ) Histograms show the luc activity obtained from zebrafish embryos microinjected simultaneously with puORFchop‐luc, phRG‐TK, and each indicated plasmid, followed by analysis of luc activity at 96 h post-fertilization (hpf). Embryos microinjected with the pCS2 vector during normal conditions (grey column) served as a control group, while the microinjected embryos at 72 hpf subjected to 40°C for 1 h comprised the heat‐shocked stress group (solid column). Relative luc activity was determined as above. *P ≤ 0.001; ***P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test.) ( D ) Western blot analysis. The protein levels of p‐eIF2α, total eIF2α, and CHOP were detected in cells overexpressing protein as indicated under either non-stress (TH, -) or stress (TH, +) conditions. The α‐tubulin and GAPDH served as internal controls. E Using quantitative RT-qPCR to determine the relative expression level of CHOP mRNA in control or HnRNPA3-overexpressing HEK293T cells under either normal (DMSO) or stress (TH) conditions. Data were averaged from three independent trials and presented as mean ± SEM. F Dual-luc assay was used to analyze the effect of HnRNPA3-knockdown on h uORF chop -MTI under either control (DMSO) or stress (TH) conditions in HEK293T cells. Data were averaged from three independent trials and presented as mean ± SEM. Statistical analysis was performed as above. G Western blot analysis. The protein levels of p‐eIF2α, total eIF2α, and CHOP expressed in HnRNPA3-knockdown cells were detected under either non-stress (TH, -) or stress (TH, +) conditions. The α‐tubulin and GAPDH served as internal controls. H RT-qPCR analyses of the gradient distribution of three mRNAs after polysome profiling assay (PPA). Lysates from control (pCS2; blank column), ENDOU-1-overexpressing (ENDOU-1; solid column), and HnRNPA3-overexpressing (HnRNPA3; grey column) cells were subjected to PPA. The resultant fractions from 1‐10 collected from a sucrose gradient were subsequently subjected to RT-qPCR assay to quantify CHOP transcripts ( CHOP mRNAs). The distribution showing the relative abundance of CHOP transcripts contained in each fraction was determined. I Relative abundance (in percentage) of CHOP mRNA presented within monosome- and polysome-containing fractions. Fractions labeled as ‘’untranslated’’ contained 40S, 60S ribosomal subunits (fractions 3 ~ 5), while fractions labeled as ‘’translated’’ contained 80 S monosome, light and heavy polysomes (fractions 6–10). Data were averaged from three independent trials and presented as mean ± SEM. *** P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test. Protein levels relative to each internal control (α-tubulin or GAPDH) are presented below each lane

Article Snippet: Antibodies were CoraLite ® Plus 488-conjugated HNRNPA3 Polyclonal antibody (Proteintech, CL488-25142; 1:200) and anti-PP11 antibody (Abcam, ab18520; 1:100) conjugated with Alexa Fluor ® 647 using Alexa Fluor ® 647 Conjugation kit (Abcam, ab269823).

Techniques: Construct, Inhibition, Control, Activity Assay, Transfection, Plasmid Preparation, Comparison, Western Blot, Quantitative RT-PCR, Expressing, Knockdown, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ENDOU-1-induced cytoplasmic HnRNPA3 recognizes m6A methylation on the upstream reading frame of human CHOP transcripts to achieve maximal CHOP translation

doi: 10.1007/s00018-026-06180-7

Figure Lengend Snippet: ENDOU-1 triggers the cytoplasmic translocation of HnRNPA3 to increase CHOP translation during ER stress. A Western blot analysis of the time course of expression levels of human ENDOU-1, HnRNPA3, and CHOP proteins in HEK293T cells after Thapsigargin (TH; stress inducer) treatment from 0〜8 h. The α‐tubulin and GAPDH served as internal controls. B Western blot analysis of HnRNPA3 in ENDOU-1-overexpressing cells or ENDOU-1-knockdown cells. The α‐tubulin served as an internal control. C Immunoblot of HnRNPA3 expression in cytoplasmic and nuclear fractions of HEK293T- or ENDOU-1-overexpressing cells. Lamin B1 and α-tubulin served as loading controls for nuclear and cytoplasmic fractions, respectively. Total protein lysates were used as an Input for the loading control. D Immunofluorescence staining of ENDOU-1 A3 (red), HnRNPA3 (green), and DAPI (blue) was obtained to observe the localization of HnRNPA3 and ENDOU-1 in HEK29ET cells under normal (DMSO) and stress conditions (TH). E Immunocytochemical detection of HnRNPA3 (green) and DAPI (blue) in HEK293T transfected with empty vector or pENDOU-1-flag. F Up: Dual-luc assay was used to analyze the effect of HnRNPA3 and its derivatives on h uORF chop -MTI under either control (DMSO) or stress (TH) conditions in HEK293T cells. Histograms presented the luc activity obtained from HEK293T cells, which were co‐transfected simultaneously with puORFchop–luc, phRG‐TK, and each of the plasmids, as indicated, for 24 h and treated with DMSO or TH for 6 h. Relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from the control group (DMSO, pCS2) normalized to 1. Data are averaged from three independent experiments and presented as mean ± SEM. *** P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test. Down: Western blot analysis. Cells in the remaining half were used to analyze the protein levels of mch-A3 derivative and CHOP, as indicated. The a-tubulin served as an internal control. Protein levels relative to each internal control (α-tubulin or GAPDH) are presented below each lane

Article Snippet: Antibodies were CoraLite ® Plus 488-conjugated HNRNPA3 Polyclonal antibody (Proteintech, CL488-25142; 1:200) and anti-PP11 antibody (Abcam, ab18520; 1:100) conjugated with Alexa Fluor ® 647 using Alexa Fluor ® 647 Conjugation kit (Abcam, ab269823).

Techniques: Translocation Assay, Western Blot, Expressing, Knockdown, Control, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Activity Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ENDOU-1-induced cytoplasmic HnRNPA3 recognizes m6A methylation on the upstream reading frame of human CHOP transcripts to achieve maximal CHOP translation

doi: 10.1007/s00018-026-06180-7

Figure Lengend Snippet: The m6A methylation on h uORF chop recognized by HnRNPA3 is necessary for allowing ENDOU-1 to achieve maximal translation of CHOP protein. A RIP-qPCR analysis showed the binding affinity of HnRNPA3 with wild-type (Wt) and mutant (AGC-mut) h uORF chop transcripts in HEK293T cells. IgG served as a negative control. Data were averaged from three independent experiments and presented as mean ± SEM. *** P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test). B Schematic representation of Wt and two m6A-2 mutated (AAU and AGC) constructs. The luc activity of HEK293T cells transfected with the indicated plasmid, under normal (DMSO) or stress (TH) conditions. The relative luc activity was represented by the fold increase of Fluc/Rluc ratio over that obtained from pCS2 transfected control group normalized as 1. Data were averaged from three independent experiments and presented as mean ± SEM. * P ≤ 0.05; *** P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test). C , D HEK293T cells were separately transfected with Wt uORF-luc ( C ) and AGC mutant-luc ( D ) plasmids for 48 h, and then the m6A level of h uORF chop and reporter DCS were evaluated using MeRIP-qPCR. Data presented in ( C , D ) were averaged from three independent experiments and are presented as mean ± SEM. *** P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test). E HepG2 cells were treated with DMSO or TH for 6 h, and then evaluated the h uORF chop m6A level was evaluated using MeRIP-qPCR. The GAPDH gene was used as a control group. F NSC34 and NSC34-SOD1G93A (mSOD1) cells were used to evaluate uORF chop m6A level using MeRIP-qPCR. The β-actin gene was used as a control. Data shown in ( E and F ) were averaged from three independent experiments and presented as mean ± SEM. *** P ≤ 0.001 (one-way ANOVA, followed by Tukey’s multiple comparison test)

Article Snippet: Antibodies were CoraLite ® Plus 488-conjugated HNRNPA3 Polyclonal antibody (Proteintech, CL488-25142; 1:200) and anti-PP11 antibody (Abcam, ab18520; 1:100) conjugated with Alexa Fluor ® 647 using Alexa Fluor ® 647 Conjugation kit (Abcam, ab269823).

Techniques: Methylation, Binding Assay, Mutagenesis, Negative Control, Comparison, Construct, Activity Assay, Transfection, Plasmid Preparation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ENDOU-1-induced cytoplasmic HnRNPA3 recognizes m6A methylation on the upstream reading frame of human CHOP transcripts to achieve maximal CHOP translation

doi: 10.1007/s00018-026-06180-7

Figure Lengend Snippet: Schematic shows how HnRNPA3 recognizes the m6A site within uORF chop and acts as a positive modulator of human CHOP mRNA translation. In the absence of stress, the methylation of uORF chop transcript is relatively low, and the translation of CHOP protein is blocked by uORF chop -MTI. In the presence of stress, (1) ENDOU-1 is increased, (2) Increased ENDOU-1 induces the highly expressed HnRNPA3, (3) Increased ENDOU-1 also induces the shift of HnRNPA3 from nucleus to cytoplasm, (4) WTAP methylates N6-adenosine site on the uORF chop transcript; (5) cytoplasmic HnRNPA3 recognizes and binds to an existing m6A methylation site on the uORF chop transcript, and, finally, (6) ENDOU-1 promotes CHOP translation by cooperating with HnRNPA3 in an m6A-dependent manner, thereby overcoming uORF chop ‐MTI. N.: Nucleus; C.: Cytoplasm

Article Snippet: Antibodies were CoraLite ® Plus 488-conjugated HNRNPA3 Polyclonal antibody (Proteintech, CL488-25142; 1:200) and anti-PP11 antibody (Abcam, ab18520; 1:100) conjugated with Alexa Fluor ® 647 using Alexa Fluor ® 647 Conjugation kit (Abcam, ab269823).

Techniques: Methylation

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Global incidence rates of multiple myeloma (MM) in males and females retrieved from the Global Burden of Disease (GBD) 2021 database. B Global mortality rates of MM in males and females from the same database. C Kaplan–Meier survival curves were generated using data from the Cancer Genome Atlas (TCGA) to compare overall survival (OS) between male and female MM patients. D Volcano plot showing differential gene expression between male and female MM patients from the GSE6401 dataset. Red dots indicate genes upregulated in males, blue dots indicate downregulated genes, and gray dots represent nonsignificant changes. E Heatmap visualizing the same dataset, with red and blue indicating male and female samples, respectively. F A heatmap showing the correlation between gene co-expression modules and clinical traits in male and female MM samples. Each cell displays the correlation coefficient and corresponding p -value between a module eigengene and a clinical trait. G A protein–protein interaction (PPI) network was constructed using STRING (v12.0) based on the intersection of differentially expressed genes and genes from the MEgrey module. Nodes represent proteins, and edges indicate predicted interactions. Hub genes were identified via maximum clique centrality scoring, suggesting their central regulatory roles. H Kaplan–Meier analysis of OS in male MM patients stratified by EIF1AY expression using the Kaplan–Meier plotter database. I Kaplan–Meier analysis of OS in male MM patients stratified by RPS4Y1 expression. J Kaplan–Meier analysis of OS in male MM patients stratified by KDM5D expression. Data were analyzed using the log-rank test. K mRNA expression levels of EIF1AY in peripheral blood samples from 30 male healthy donors (HDs) and 54 male patients. L mRNA expression levels of RPS4Y1 in the same cohort. M Protein expression levels of RPS4Y1 and EIF1AY in HDs and MM patients. N Dual-color fluorescence in situ hybridization (FISH) targeting EIF1AY (Yq11.223, orange) and centromere X (CenX, green) was performed on bone marrow cells from HDs and MM patients. O Representative images show the presence of EIF1AY-positive cells in HDs and loss of EIF1AY in MM samples.

Article Snippet: Primary antibodies included GAPDH (10494-1-AP), beta tubulin (10094-1-AP), EIF1AY (11193-1-AP), RPS4Y1 (17296-1-AP), CD134 (20006-1-AP), and secondary antibody (RGAR004) (all from Proteintech).

Techniques: Generated, Gene Expression, Expressing, Construct, Fluorescence, In Situ Hybridization

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Cell proliferation assay showing the growth rate of RPMI 8226 cells overexpressing EIF1AY. B Tumor weight of male MM xenografts following EIF1AY overexpression. C Representative images of xenograft tumors from the same experiment. D Tumor growth curves of xenografts over 30 days. E Hematoxylin and eosin (HE) staining and immunohistochemical (IHC) detection of EIF1AY and Ki-67 expression in xenograft tissues. Scale bar: 80 μm. F Comparative analysis of the fractional distribution of 22 immune cell types in male versus female MM samples from the GSE164701 dataset. G Phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells were differentiated into M0 macrophages (THP-1-Mφ). qRT-PCR analysis of M2 macrophage markers (IL-10, TGF-β, ARG1, and CD206) in THP-1-Mφ exposed to conditioned medium (CM) from EIF1AY-overexpressing MM cells. H CD206 protein expression in THP-1-Mφ treated with CM from EIF1AY-overexpressing MM cells was evaluated by Western blot analysis. I Flow cytometric analysis of CD206 expression in THP-1-Mφ across the indicated groups. J IHC analysis of CD206 expression in xenograft tumor tissues. Scale bar: 80 μm. K Chemotaxis assay assessing migration of THP-1-Mφ in response to CM from EIF1AY-overexpressing U266 cells. L Chemotaxis assay assessing migration of THP-1-Mφ in response to CM from EIF1AY-overexpressing RPMI-8226 cells under the same experimental conditions. M Proliferation of MM cells cultured with CM from M2-polarized THP-1-Mφ induced by EIF1AY-overexpressing U266 cells. N Proliferation of MM cells cultured with CM from M2-polarized THP-1-Mφ induced by EIF1AY-overexpressing RPMI-8226 cells under the same experimental conditions.

Article Snippet: Primary antibodies included GAPDH (10494-1-AP), beta tubulin (10094-1-AP), EIF1AY (11193-1-AP), RPS4Y1 (17296-1-AP), CD134 (20006-1-AP), and secondary antibody (RGAR004) (all from Proteintech).

Techniques: Proliferation Assay, Over Expression, Staining, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Western Blot, Chemotaxis Assay, Migration, Cell Culture

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Volcano plot of differentially expressed genes between normal macrophages (MΦs) and tumor-associated macrophages (TAMs) from GSE143583 . B Heatmap of the 381 differentially expressed genes between MΦs and TAMs; color intensity indicates expression. C WGCNA module-trait correlation heatmap; rows = module eigengenes, columns = macrophage subtypes. D Venn diagram showing overlap among differentially expressed genes, MEturquoise module genes, and membrane proteins from the Membranome database. E PPI network of genes in ( D ) generated via STRING; edges indicate predicted interactions. F DDR1 expression levels in MΦs and TAMs from GSE143583 . G DDR1 mRNA in THP-1-Mφ cultured with or without CM from MM cells, measured by qRT-PCR. H qRT-PCR analysis of IL-10, TGF-β, ARG1, and CD206 mRNA levels in THP-1-Mφ cultured with CM from MM cells overexpressing DDR1. I Flow cytometry analysis of CD206 expression in the indicated experimental groups. J qRT-PCR analysis of DDR1 mRNA expression in THP-1-Mφ cultured with or without CM from EIF1AY-overexpressing RPMI-8226 cells. K qRT-PCR analysis of DDR1 mRNA expression in THP-1-Mφ cultured with or without CM from EIF1AY-overexpressing U266 cells under the same experimental conditions. L Immunofluorescence (IF) analysis of DDR1 expression in THP-1-Mφ cultured with or without CM from EIF1AY-overexpressing MM cells. M Migration assay of THP-1-Mφ transduced to overexpress DDR1, cultured in CM from MM cells with or without EIF1AY overexpression (CM Vector or CM oe-EIF1AY). Recruited cells in the lower chamber were quantified to assess whether DDR1 overexpression in THP-1-Mφ rescues the migration impairment induced by EIF1AY expression in MM cells. N THP-1-Mφ pretreated with CM from EIF1AY-overexpressing or control MM cells were subsequently transduced with DDR1 and indirectly cocultured with MM cells in a transwell system. MM cell proliferation was then assessed to evaluate the effect of macrophage-derived signals. O qRT-PCR analysis of IL-4 and IL-13 mRNA levels in RPMI 8226 cells following EIF1AY overexpression. P ELISA analysis of IL-4 and IL-13 secretion in culture supernatants from EIF1AY-overexpressing MM cells. Q qRT-PCR analysis of DDR1 mRNA expression in THP-1-Mφ stimulated with or without IL-4 and IL-13.

Article Snippet: Primary antibodies included GAPDH (10494-1-AP), beta tubulin (10094-1-AP), EIF1AY (11193-1-AP), RPS4Y1 (17296-1-AP), CD134 (20006-1-AP), and secondary antibody (RGAR004) (all from Proteintech).

Techniques: Expressing, Membrane, Generated, Cell Culture, Quantitative RT-PCR, Flow Cytometry, Immunofluorescence, Migration, Over Expression, Plasmid Preparation, Control, Transduction, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Scatterplot showing positive correlation between RPS4Y1 and EIF1AY in the GSE6401 dataset. B Correlation validated in male MM patient samples. C qRT-PCR of EIF1AY mRNA in RPMI-8226 cells after RPS4Y1 knockdown. D qRT-PCR of EIF1AY mRNA in RPMI-8226 cells after RPS4Y1 overexpression. E Western blot of EIF1AY protein in RPMI-8226 cells after RPS4Y1 knockdown. F Western blot of EIF1AY protein in RPMI-8226 cells after RPS4Y1 overexpression. G qRT-PCR of IL-10, TGF-β, ARG1, and CD206 in THP-1-Mφ cultured with CM from RPS4Y1-overexpressing MM cells. H Western blot of CD206 in THP-1-Mφ treated as in ( G ). I qRT-PCR of DDR1 mRNA in THP-1-Mφ cultured with CM from RPS4Y1-overexpressing RPMI-8226 cells. J IF staining of DDR1 in RPS4Y1-overexpressing RPMI-8226 cells. K qRT-PCR of IL-4 mRNA in RPMI-8226 cells overexpressing RPS4Y1. L qRT-PCR of IL-13 mRNA in RPMI-8226 cells treated as in ( K ). M ELISA quantification of IL-4 in culture supernatants after RPS4Y1 overexpression. N ELISA quantification of IL-13 in culture supernatants after RPS4Y1 overexpression. O Structure-based modeling of the RPS4Y1–EIF1AY complex showing key interaction residues and predicted binding affinity. P Co-IP in RPMI-8226 cells with anti-EIF1AY to detect RPS4Y1. Q Co-IP in RPMI-8226 cells with anti-RPS4Y1 to detect EIF1AY. R Co-IP in U266 cells with anti-EIF1AY to detect RPS4Y1. S Co-IP in U266 cells with anti-RPS4Y1 to detect EIF1AY. T Co-IP in Flag-RPS4Y1 cells to detect EIF1AY. U Co-IP in Flag-EIF1AY cells to detect RPS4Y1. V THP-1-Mφ chemotactic migration assays were performed using CM derived from RPS4Y1-overexpressing MM cells, with or without EIF1AY knockdown. W The number of tumor cells was quantified following incubation with CM from polarized THP-1-Mφ induced by either RPS4Y1-overexpressing MM cells or RPS4Y1-overexpressing MM cells with EIF1AY knockdown.

Article Snippet: Primary antibodies included GAPDH (10494-1-AP), beta tubulin (10094-1-AP), EIF1AY (11193-1-AP), RPS4Y1 (17296-1-AP), CD134 (20006-1-AP), and secondary antibody (RGAR004) (all from Proteintech).

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Co-Immunoprecipitation Assay, Migration, Derivative Assay, Incubation

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: A Correlation between CD134 and RPS4Y1 expression in male MM samples. B Correlation between CD134 and EIF1AY expression in male MM samples. C qRT-PCR analysis of CD134 mRNA in RPMI-8226 cells after RPS4Y1 knockdown. D qRT-PCR analysis of CD134 mRNA in RPMI-8226 cells after RPS4Y1 overexpression. E qRT-PCR analysis of CD134 mRNA in RPMI-8226 cells after EIF1AY knockdown. F qRT-PCR analysis of CD134 mRNA in RPMI-8226 cells after EIF1AY overexpression. G – J Corresponding protein expression levels of CD134 in RPMI 8226 cells upon RPS4Y1 or EIF1AY knockdown and overexpression. G Western blot analysis of CD134 protein in RPMI-8226 cells after RPS4Y1 knockdown. H Western blot analysis of CD134 protein in RPMI-8226 cells after RPS4Y1 overexpression. I Western blot analysis of CD134 protein in RPMI-8226 cells after EIF1AY knockdown. J Western blot analysis of CD134 protein in RPMI-8226 cells after EIF1AY overexpression. K CD134 protein expression in RPMI 8226 cells was analyzed by Western blot following overexpression of RPS4Y1 alone or combined overexpression of RPS4Y1 and shEIF1AY. L Chemotactic migration of THP-1-Mφ in response to CM derived from MM cells treated with siCD134, oe-RPS4Y1 + siCD134, or oe-EIF1AY + siCD134. M Quantification of MM cell numbers cultured with CM derived from THP-1-Mφ polarized using the same treatment conditions described in ( L ). N RNA pull-down assay in RPMI-8226 cells using CD134-specific probes to detect binding to RPS4Y1 and EIF1AY. O RNA pull-down assay in U266 cells using CD134-specific probes to detect binding to RPS4Y1 and EIF1AY. P RNA immunoprecipitation (RIP) assay in Flag-RPS4Y1–overexpressing RPMI-8226 and U266 cells using anti-Flag antibodies. CD134 mRNA enrichment was quantified by qRT-PCR relative to IgG control (2 −ΔΔCt ). Q RIP assay in Flag-EIF1AY-overexpressing RPMI-8226 and U266 cells using anti-Flag antibodies. R CD134 mRNA stability in RPMI-8226 cells following RPS4Y1 knockdown, assessed by actinomycin D chase and qRT-PCR. S CD134 mRNA stability in RPMI-8226 cells following EIF1AY knockdown, assessed by actinomycin D chase and qRT-PCR.

Article Snippet: Primary antibodies included GAPDH (10494-1-AP), beta tubulin (10094-1-AP), EIF1AY (11193-1-AP), RPS4Y1 (17296-1-AP), CD134 (20006-1-AP), and secondary antibody (RGAR004) (all from Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Migration, Derivative Assay, Cell Culture, Pull Down Assay, Binding Assay, RNA Immunoprecipitation, Control

Journal: NPJ Precision Oncology

Article Title: Y chromosome-linked EIF1AY deletion drives sex differences in multiple myeloma

doi: 10.1038/s41698-026-01317-0

Figure Lengend Snippet: EIF1AY, a Y chromosome-encoded protein, forms a complex with RPS4Y1 to directly bind and stabilize CD134 mRNA, thereby sustaining CD134 expression in MM cells. CD134 signaling suppresses the secretion of IL-4 and IL-13, cytokines that normally induce DDR1 expression on macrophages and promote their polarization toward the tumor-supportive M2 phenotype. The RPS4Y1-EIF1AY-CD134 axis inhibits M2 macrophage polarization and recruitment, limiting MM cell proliferation. Loss of EIF1AY disrupts this axis, resulting in increased IL-4 and IL-13 secretion, upregulated DDR1 expression on macrophages, enhanced M2 polarization, and accelerated tumor progression. This feed-forward regulatory loop reveals a Y chromosome-linked immune mechanism underlying sex differences in MM and identifies EIF1AY as a potential target for precision immunotherapy in male patients.

Article Snippet: Primary antibodies included GAPDH (10494-1-AP), beta tubulin (10094-1-AP), EIF1AY (11193-1-AP), RPS4Y1 (17296-1-AP), CD134 (20006-1-AP), and secondary antibody (RGAR004) (all from Proteintech).

Techniques: Expressing

Journal: Advanced Science

Article Title: Astrocytic PCBP1 Suppresses Ferroptosis to Restore Glutamatergic Homeostasis and Mitigate Stress‐Induced Depression in Male Mice

doi: 10.1002/advs.202513438

Figure Lengend Snippet: Astrocytic PCBP1 enhances glutamatergic neuronal activity and neurophysiology. a) c‐Fos expression in vHip of mice following CUMS. Above: Representative images and quantification of c‐Fos‐positive glutamatergic neurons. Below: Representative images and quantification of c‐Fos‐positive GABAergic neurons. Scale bars = 50 µm. n = 9 slices from 3 animals per group. b) Timeline of fiber photometry recording Ca 2+ and glutamate signals from vHip c) Left: Representative images validate GCaMP6s expression in vHip. Scale bar = 500 µm. Right: Representative images showing the overlap between GCaMP6s‐expressing cells (green) and glutamatergic neurons (violet). Scale bar = 50 µm. d) Representative images of c‐Fos‐positive glutamatergic neurons. Scale bars = 100 or 20 µm. e) Statistical analysis of c‐Fos co‐labeled glutamatergic neuron in vHip of mice. f) Schematic of fiber photometry recording in TST and representative Ca 2+ signals photometric traces in response to the TST during struggling and immobility phases. Scale bars = 10 s. g) Ca 2 ⁺ signal recording in vHip during TST. Left: Representative heatmaps showing Ca 2 ⁺ signal in response to TST during struggling. Right: Z‐score traces and quantification of average Z‐scores during TST. n = 5 mice per group. h) Representative images of the AAV vectors engineered to express the glutamate sensor iGluSnFR under the hSyn promoter in the vHip. Scale bars = 500 µm / 50 µm. i) Representative trace of glutamate signal event detection during TST. Red dots indicate detected events. j) Event‐based quantification. Event frequency (left), AUC (middle) and decay tau (right). n = 5 mice per group. k) Timeline of electrophysiological recordings from vHip glutamatergic neurons. l) Quantification of mEPSC frequency (left) and amplitude (right) recorded from vHip glutamatergic neurons. n = 10–12 cells from 3 mice per group. m) Left: Evoked firing rates of action potentials recorded from eGFP⁺ neurons in the vHip. Right: Quantifications of rheobase and resting membrane potential recorded from eGFP + neurons in vHip. n = 10 cells from 3 mice per group. Statistical analyses included unpaired t‐test, one‐way/two‐way ANOVA followed by Tukey's post hoc test and Kruskal‐Wallis test. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For immunofluorescence analysis, the 30‐μm‐thick sections were incubated overnight at 4 °C with primary antibodies against NEUN (94403S, Cell Signaling Technology), GFAP (GB11096, Servicebio), IBA‐1 (GB12105, Servicebio), CaMKII (11533‐1‐AP, Proteintech), GAD67 (PA5‐21397, Invitrogen), c‐Fos (OB‐PGP080, Oasis biogarm), GPX4 (ab125066, Abcam), and PCBP1 (14523‐1‐AP, Proteintech).

Techniques: Activity Assay, Expressing, Labeling, Membrane