Journal: Oncology Reports

Article Title: NR4A1 mediates chemotherapy-induced senescence via the PI3K/AKT pathway in gastric cancer cells

doi: 10.3892/or.2026.9080

Figure Lengend Snippet: OXA induces senescence was verified in gastric cancer cells. (A and B) SA-β-Gal staining of Ctrl and OXA groups and quantification of SA-β-Gal positive cells in AGS and MKN45 cells. (C) p21 protein expression and quantification. (D and E) Immunofluorescence of γ-H2AX and quantification. Red fluorescence indicates γ-H2AX staining, and blue fluorescence reflects nuclear staining with DAPI. (F) mRNA levels of the senescence-associated secretory phenotype factors. (G) CDK4, cyclin D1, RB, p-RB protein expression, and quantification in the two groups. Data represent the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. OXA, oxaliplatin; SA-β-Gal, senescence-associated β-galactosidase; p-, phosphorylated.

Article Snippet: AGS and MKN45 human GC cell lines were acquired from the Procell Life Science & Technology Co., Ltd. and maintained in RPMI-1640 containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO 2 condition.

Techniques: Staining, Expressing, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

doi: 10.64898/2026.03.26.714452

Figure Lengend Snippet: A) RNA immunoprecipitation (RIP) from GC-2spc cells using SMG6 and PIWIL1 antibodies. Rabbit IgG was used as a negative control. Left panel shows Western blotting (WB) of SMG6 and PIWIL1. Right panel shows RT-PCR after IP using Ccny and Taf1d specific primers. B) Validation of the knockdown efficiency after transfection of small interfering RNA (siRNA) for Smg6 or Piwil1 (si-Smg6, si-Piwil1) in GC-2spd cells by RT-qPCR using Smg6 or Piwil1 primers. Scrambled siRNA was used as a negative control (si-Scr). C) Validation of the overexpression of SMG6 or PIWIL1 in GC-2spd cells using HA-SMG6 or FLAG-PIWIL1 expression plasmids, respectively. Empty pcdna3.1 vector as a control. D) RT-qPCR using Ccny and Taf1d -specific primers after siRNA-mediated downregulation of Smg6 or Piwil1 in GC-2spd cells. E) RT-qPCR using Ccny and Taf1d -specific primers after overexpression of HA-SMG6 or FLAG-PIWIL1 in GC-2spd cells. F) Overexpression of SMG6 (HA-SMG6) in GC-2spd cells in the presence or absence of Piwil1 knockdown by siRNA. RT-qPCR analysis of Ccny and Taf1d mRNAs was performed to assess whether PIWIL1 is required for SMG6-mediated regulation of these targets. G) Control (si-Scr) or PIWIL1 knockdown (si-Piwil1) GC-2spd cells were subjected to SMG6 RIP, followed by RT-qPCR to assess the association of SMG6 with Ccny and Taf1d mRNAs. Taf1d and Ccny transcripts were quantified and normalized to input RNA. Data represent mean ± S.D. from three independent experiments, *P < 0.05. H) Western blotting with anti-SMG6 antibody to validate equal IP of SMG6 from control (si-Scr) and Piwil1 -silenced (si- Piwil1) cells. Two representative IPs (RIP-1, RIP-2) are shown for both conditions. For all graphs (B-F), means of three independent experiments are shown, and Rpl19 was used as a reference gene. Data represent mean ± S.D. from three independent experiments, *P < 0.05.

Article Snippet: Mouse spermatogonial GC-2spd cells (ATCC® CRL-2196TM, USA) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, D-18900; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, S1810; Biowest) and 1% penicillin–streptomycin (15140-122; Gibco).

Techniques: RNA Immunoprecipitation, Negative Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Knockdown, Transfection, Small Interfering RNA, Quantitative RT-PCR, Over Expression, Expressing, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

doi: 10.64898/2026.03.26.714452

Figure Lengend Snippet: A) m⁶A -RIP followed by RT-PCR using primers for Taf1d and Ccny. Mouse IgG was used as a control. B) METTL3 knockdown by siRNA (si-Mettl3) followed by RT-PCR to amplify Ccny and Taf1d mRNAs. Scrambled siRNA (si-Scr) was used as a control. C) Downregulation of METTL3 was validated by anti-METTL3 western blotting. Antibody against ACTIN was used as a loading control. D) GC-2spd cells were treated with the methyltransferase inhibitor STC-15 followed by RT-PCR using Ccny and Taf1d primers (left panel). Right panel shows the levels of m⁶A RNA in control (DMSO) and STC-15-treated cells by dot blotting with anti-m6A antibody (two independent replicates are shown). E) In situ hybridization using Ccny -specific probe of DMSO- or STC-15 treated GC-2spd cells. DAPI stains the nuclei (grey). Scale bar: 10 µm. F) GC-2spd cells were transfected with control siRNA (si-Scr), Smg6 - or Piwil1 -targeting siRNA (si-Smg6 or si-Piwil1), empty pcdna3.1 plasmid (control) or HA-SMG6 and FLAG-PIWIL1 overexpressing plasmids before m⁶A - RIP. m6-A-modified Taf1d was detected with RT-PCR from anti-m⁶A RIP samples. Mouse IgG was used as a control in the RIP. G) GC-2spd cells were treated with DMSO, METTL3a1, or STC-15 before anti-SMG6-RIP. SMG6-associated Ccny and Taf1d mRNAs were detected with RT-PCR. Rabbit IgG was used as a control in the RIP. H) Western blot with anti-SMG6 antibody to validate equal IP of SMG6 from DMSO, STC-15, and METTL3a1 treated cells. One representative RIP experiment is shown for each condition.

Article Snippet: Mouse spermatogonial GC-2spd cells (ATCC® CRL-2196TM, USA) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, D-18900; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, S1810; Biowest) and 1% penicillin–streptomycin (15140-122; Gibco).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Western Blot, In Situ Hybridization, Transfection, Plasmid Preparation, Modification

Journal: bioRxiv

Article Title: Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

doi: 10.64898/2026.03.26.714452

Figure Lengend Snippet: A) Immunoprecipitation of PIWIL1 and SMG6 from adult mouse testes. Successful IP was confirmed with Western blotting (WB) using SMG6 and PIWIL1 antibodies. Associated small RNAs were visualized in the UREA-PAGE gel with SYBR Gold staining, revealing a 26-32-nt population consistent with piRNAs length. B) Transfection of PIWIL1-associated testis RNAs into GC2spd cells, followed by RT-qPCR analysis of Ccny target mRNA. Gapdh was used as a reference gene. Data represent mean ± S.D. from two independent experiments. C) Transfection of Ccny -targeting piRNA ( pi-Ccny ) induces Ccny mRNA downregulation in 3T3 cells only when PIWIL1 is overexpressed. Scrambled piRNA ( pi-Scr ) was used as a control for Ccny -targeting piRNA. Empty pcdna3.1. vector (control) was used as a control for FLAG-PIWIL1 overexpression. D) GC-2spd cells were transfected with pi-Scr or pi-Ccny , followed by RT-qPCR using Ccny primers at different time points after transfection to monitor Ccny mRNA levels. E) GC-2spd cells were subjected to siRNA-mediated knockdown of Smg6 prior to transfection with the synthetic pi-Ccny , followed by RT-PCR analysis of Ccny mRNA. F) Same experiment as in (E), but Ccny expression was detected by RT-qPCR. G) GC-2spd cells were treated with the METTL3 inhibitor STC-15 to block m⁶A deposition, followed by transfection with the synthetic pi-Ccny and RT-PCR analysis of Ccny mRNA. For graphs (D,F), Gapdh was used as a reference gene. Data represent mean ± S.D. from three independent experiments; *P < 0.05.

Article Snippet: Mouse spermatogonial GC-2spd cells (ATCC® CRL-2196TM, USA) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, D-18900; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, S1810; Biowest) and 1% penicillin–streptomycin (15140-122; Gibco).

Techniques: Immunoprecipitation, Western Blot, Staining, Transfection, Quantitative RT-PCR, Control, Plasmid Preparation, Over Expression, Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Blocking Assay

Journal: eLife

Article Title: Blocking SHP2 benefits FGFR2 inhibitor and overcomes its resistance in FGFR2 -amplified gastric cancer

doi: 10.7554/eLife.104060

Figure Lengend Snippet: Sensitivity of KATOIII and SNU-16 to ( A ) SHP099, ( B ) AZD4547, or combination therapy with different concentration gradients (n=4; two-way ANOVA). Effects of different treatments on cell apoptosis of ( C ) KATOIII and ( D ) SNU-16 after 48-hour drugs incubation (n=3; one-way ANOVA). ( E ) KATOIII and ( F ) SNU-16 were incubated with vehicle, SHP099 10 μM, AZD4547 3 nM or combination therapies for 1 hour or 48 hours, then cell lysates were immunoblotted for phospho-FGFR and total-FGFR2, phospho-SHP2 and total-SHP2, phospho-Erk and total-Erk, phospho-p38 and total-p38, phospho-AKT and total-AKT, and phospho-mTOR and total-mTOR. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. p-Values are determined by ordinary one-way ANOVA or two-way ANOVA. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 2—source data 2. Original file for western blots displayed in .

Article Snippet: Human GC cell lines SNU-16 (ATCC CRL-5974), MKN45 (KANGBAI CBP60488), NUGC4 (KANGBAI CBP60493), HGC27 (KANGBAI CBP60480), and SNU601 (KANGBAI CBP60507) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: Concentration Assay, Incubation, Western Blot

Journal: eLife

Article Title: Blocking SHP2 benefits FGFR2 inhibitor and overcomes its resistance in FGFR2 -amplified gastric cancer

doi: 10.7554/eLife.104060

Figure Lengend Snippet: BALB/c nude mice were injected with 1×10 7 SNU-16 cancer cells and received different formulations by oral gavage every day upon tumor volumes reached 100–150 mm 3 . ( A ) Schematic of SHP099 and AZD4547 therapeutic schedule in FGFR2 -amplified SNU-16 subcutaneous xenograft model. ( B ) Representative images of tumors (n=5). ( C ) Tumor volumes (n=5; two-way ANOVA), ( D ) tumor weights (n=5; one-way ANOVA) and ( E ) body weights (n=5; two-way ANOVA) of different groups. ( F ) Tumors were harvested 6 hours after the last dose of drugs, and cell lysates from tumor tissues were immunoblotted for phospho-FGFR and total-FGFR2, phospho-SHP2 and total-SHP2, phospho-Erk and total-Erk, phospho-AKT and total-AKT, and phospho-mTOR and total-mTOR. Data are shown as mean ± SEM. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. p-Values are determined by ordinary one-way ANOVA or two-way ANOVA. Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 4—source data 2. Original file for western blots displayed in .

Article Snippet: Human GC cell lines SNU-16 (ATCC CRL-5974), MKN45 (KANGBAI CBP60488), NUGC4 (KANGBAI CBP60493), HGC27 (KANGBAI CBP60480), and SNU601 (KANGBAI CBP60507) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: Injection, Amplification, Western Blot

Journal: eLife

Article Title: Blocking SHP2 benefits FGFR2 inhibitor and overcomes its resistance in FGFR2 -amplified gastric cancer

doi: 10.7554/eLife.104060

Figure Lengend Snippet: ( A ) Overview of FGFR2 alterations individually in GC patients from Nanjing Drum Tower hospital cohort. ( B ) PD-L1 mRNA expression levels were analyzed between FGFR2 -amplified group (n=13) and FGFR2 -unamplified group (n=250) from TCGA-STAD cohort (Welch’s t -test). ( C ) Proportions of MSI-H or MSS in FGFR2 -amplified group (n=12) and FGFR2 -unamplified group (n=237) from TCGA-STAD cohort (Fisher’s exact test). Human peripheral blood mononuclear cells (PBMCs) were incubated with different administrations in the presence of 0.25 μg/ml human anti-CD3 and 1 μg/ml human anti-CD28. Proportions of ( D ) IFN-γ/CD8+ cells were detected by flow cytometry assay after 24 hours of drugs incubation (n=3; one-way ANOVA). ( E ) Expression levels of IFN-γ in cellular supernatant were measured by Cytometric Bead Array (CBA) assay after 24 hours of drugs incubation (n=3; one-way ANOVA). ( F ) Cell viability of human PBMCs after 48 hours of drugs incubation (n=4; one-way ANOVA). ( G, H ) After 48 hours of advance drugs stimulation in human PBMCs, cytotoxicities of human PBMCs against SNU-16 at different E:T ratios of 10:1, 20:1, 40:1 were analyzed by flow cytometry assay after CFSE/PI staining (n=3; two-way ANOVA). Data are shown as mean ± SEM. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. p-Values are determined by Welch’s t -test, Fisher’s exact test, ordinary one-way ANOVA or two-way ANOVA.

Article Snippet: Human GC cell lines SNU-16 (ATCC CRL-5974), MKN45 (KANGBAI CBP60488), NUGC4 (KANGBAI CBP60493), HGC27 (KANGBAI CBP60480), and SNU601 (KANGBAI CBP60507) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Techniques: Expressing, Amplification, Incubation, Flow Cytometry, Staining