Journal: bioRxiv

Article Title: Peripheral extracellular vesicle-derived miR-150-3p exacerbates acute kidney injury following acute pancreatitis by promoting ferroptosis through FTH1 signaling

doi: 10.1101/2023.01.17.524353

Figure Lengend Snippet: [A] Representative renal electron microscopy photographs of kidney mitochondria harvested from rats that were subjected to AP-AKI, Ctrl-ev and AP-AKI-ev after 24 h AP models were established. Mitochondrial crumpling and higher membrane density are shown (white arrow). Scale bar=500 nm. [B] The tissue levels of Fe 2+ , GSH and MDA in rats as described above. [C] Representative fluorescent photographs of GPX4 in kidneys harvested from rats as described above. The distribution of GPX4 (left) and the intensity level of GPX4 (right) are shown (red, GPX4). Scale bar=100 μm. [D] Representative western blot images and quantifications of GPX4 and Cox-2 in the kidney as described above. β-actin was used as the protein loading control. Data are plotted as the mean ± SEM. P values were determined by a one-way ANOVA; n.s., no significance, *P < 0.05, **P < 0.01. AP-AKI, acute kidney injury following acute pancreatitis; GSH, glutathione; MDA, malondialdehyde; GPX4, glutathione peroxidase 4; COX-2, cyclooxygenase 2 .

Article Snippet: The membrane was then blocked with 5% skim milk at 37 °C for 1 h and incubated overnight with the following antibodies: anti-Alix (1:1,000, ab275377, Abcam, UK), anti-TSG101 (1:1,000, ab125011, Abcam), anti-CD63 (1:1,000, A5271, ABclonal), anti-GPX4 (1:1,000, C29H4, Cell Signaling Technology), anti-Cox-2 (1:1,000, 9461, Cell Signaling Technology), anti-β-actin (1:2,000, BA2305, Boster), and anti-FTH1 (1:1,000, BA0362, Boster).

Techniques: Electron Microscopy, Western Blot

Journal: bioRxiv

Article Title: Peripheral extracellular vesicle-derived miR-150-3p exacerbates acute kidney injury following acute pancreatitis by promoting ferroptosis through FTH1 signaling

doi: 10.1101/2023.01.17.524353

Figure Lengend Snippet: [A] Representative fluorescent images indicative of normal and AP-AKI-derived EVs and differences in HK-2 cells (green, EVs). Scale bar=30 μm. [B] Relative Fe2+ levels between the NC-mimic-, miR-150-3P-mimic-, NC-inhibitor- and miR-150-3P-inhibitor-transfected groups. The expression levels of miR-150-3P in the NC-mimic, miR-150-3P-mimic, NC-inhibitor and miR-150-3P-inhibitor groups (left). Fe2+ levels between the four groups (right). [C, D] Representative western blot images and quantifications of GPX4 and Cox-2 protein expression in HK-2 cells as described above. β-actin was used as the protein loading control. [E] Representative flow cytometry results for cell lipid peroxidation as described above. [F] Representative fluorescent images indicative of MTP by JC-1 staining in the cells mentioned above; the ratio of red/green fluorescence was calculated to indicate MTP. Scale bar=30 μm. Data are plotted as the mean ± SEM (n=4). P values were determined by a one-way ANOVA; n.s., no significance, *P < 0.05, **P < 0.01. GPX4, glutathione peroxidase 4; COX-2, cyclooxygenase 2; MTP, mitochondrial membrane potential .

Article Snippet: The membrane was then blocked with 5% skim milk at 37 °C for 1 h and incubated overnight with the following antibodies: anti-Alix (1:1,000, ab275377, Abcam, UK), anti-TSG101 (1:1,000, ab125011, Abcam), anti-CD63 (1:1,000, A5271, ABclonal), anti-GPX4 (1:1,000, C29H4, Cell Signaling Technology), anti-Cox-2 (1:1,000, 9461, Cell Signaling Technology), anti-β-actin (1:2,000, BA2305, Boster), and anti-FTH1 (1:1,000, BA0362, Boster).

Techniques: Derivative Assay, Transfection, Expressing, Western Blot, Flow Cytometry, Staining, Fluorescence

Journal: bioRxiv

Article Title: Peripheral extracellular vesicle-derived miR-150-3p exacerbates acute kidney injury following acute pancreatitis by promoting ferroptosis through FTH1 signaling

doi: 10.1101/2023.01.17.524353

Figure Lengend Snippet: [A] Three online analysis tools predicted 102 overlapping downstream genes of miR-150-3P. [B] TargetScan results show that the 3’UTR of FTH1 contains a complementary binding site for miR-150-3p. [C] Relative dual luciferase activities of the reporter between 3’UTR-NC, 3’UTR, 3’UTR-mut and miR-150-3p. [D, E] Representative western blot images and quantifications of GPX4 and Cox-2 protein expressions in HK-2 cells treated separately with negative control inhibitor, negative control siRNA, siRNA equipped with ferritin heavy chain 1 and miR-150-3p inhibitor. [F] Representative fluorescent photographs of FTH1 in HK-2 cells transfected with negative control inhibitor, miR-150-3P inhibitor, negative control mimic and miR-150-3P mimic. Images show FTH1 (green) and nuclei (blue). [G] Representative western blot images and quantifications of COX-2 and GPX4 protein expressions. β-actin was used as the protein loading control. Scale bar=30 μm. The left histogram shows the fluorescence intensity between the four groups. Data are plotted as the mean ± SEM (n=4). P values were determined by a one-way ANOVA; n.s., no significance, *P < 0.05, **P < 0.01. GPX4, glutathione peroxidase 4; COX-2, cyclooxygenase 2 .

Article Snippet: The membrane was then blocked with 5% skim milk at 37 °C for 1 h and incubated overnight with the following antibodies: anti-Alix (1:1,000, ab275377, Abcam, UK), anti-TSG101 (1:1,000, ab125011, Abcam), anti-CD63 (1:1,000, A5271, ABclonal), anti-GPX4 (1:1,000, C29H4, Cell Signaling Technology), anti-Cox-2 (1:1,000, 9461, Cell Signaling Technology), anti-β-actin (1:2,000, BA2305, Boster), and anti-FTH1 (1:1,000, BA0362, Boster).

Techniques: Binding Assay, Luciferase, Western Blot, Negative Control, Transfection, Fluorescence

Journal: bioRxiv

Article Title: Peripheral extracellular vesicle-derived miR-150-3p exacerbates acute kidney injury following acute pancreatitis by promoting ferroptosis through FTH1 signaling

doi: 10.1101/2023.01.17.524353

Figure Lengend Snippet: [A, B] Representative fluorescence photographs of Cox-2 and GPX4 in HK-2 cells transfected with miR-150-3P inhibitor, siFTH1 and miR-150-3P inhibitor-associated siFTH1. Images show Cox-2 and GPX4 (green) and nuclei (blue). [C] Representative fluorescent images indicative of MTP by JC-1 staining in the cells mentioned above; the ratio of red/green fluorescence was calculated to indicate MTP. [D] Representative western blot images and quantifications of COX-2 and GPX4 protein expressions. β-actin was used as the protein loading control. Scale bar=100 μm. Data are plotted as the mean ± SEM (n=4). P values were determined by a one-way ANOVA; n.s., no significance, *P < 0.05, **P < 0.01. GPX4, glutathione peroxidase 4; Cox-2, cyclooxygenase 2; MTP, mitochondrial membrane potential .

Article Snippet: The membrane was then blocked with 5% skim milk at 37 °C for 1 h and incubated overnight with the following antibodies: anti-Alix (1:1,000, ab275377, Abcam, UK), anti-TSG101 (1:1,000, ab125011, Abcam), anti-CD63 (1:1,000, A5271, ABclonal), anti-GPX4 (1:1,000, C29H4, Cell Signaling Technology), anti-Cox-2 (1:1,000, 9461, Cell Signaling Technology), anti-β-actin (1:2,000, BA2305, Boster), and anti-FTH1 (1:1,000, BA0362, Boster).

Techniques: Fluorescence, Transfection, Staining, Western Blot

Journal: bioRxiv

Article Title: Peripheral extracellular vesicle-derived miR-150-3p exacerbates acute kidney injury following acute pancreatitis by promoting ferroptosis through FTH1 signaling

doi: 10.1101/2023.01.17.524353

Figure Lengend Snippet: [A] The kidney miR-150-3p, GSH, MDA, blood serum creatine and blood urea nitrogen levels were determined and hemospasia from rats that were subjected to negative control agomir, miR-150-3p agomir, negative control antagomir and miR-150-3p antagomir 24 h after the AP model was produced. [B] Representative photographs and histological scores of HE-stained kidney sections harvested from rats as mentioned above. Scale bar=100 μm. [C] Representative renal electron microscopy photographs of kidney mitochondria harvested from rats that were mentioned above. Mitochondrial crumpling and higher membrane density are shown (white arrow). Scale Bar=500nm. [D] Representative western blot images and quantifications of FTH1, GPX4 and Cox-2 proteins between the four groups. [E] Representative fluorescent photographs of GPX4 (above) and FTH1 (below) in kidney sections harvested from rats as described above. Imagines of GPX4 and FTH1 are shown (red). Scale bar=100 μm. Data are plotted as the mean ± SEM (n=4). P values were determined by a one-way ANOVA; n.s., no significance, *P < 0.05, **P < 0.01. GSH, glutathione; MDA, malondialdehyde; GPX4, glutathione peroxidase 4; COX-2, cyclooxygenase 2; FTH1, ferritin heavy chain 1p; H&E stain, hematoxylin-eosin staining; IF, immunofluorescence .

Article Snippet: The membrane was then blocked with 5% skim milk at 37 °C for 1 h and incubated overnight with the following antibodies: anti-Alix (1:1,000, ab275377, Abcam, UK), anti-TSG101 (1:1,000, ab125011, Abcam), anti-CD63 (1:1,000, A5271, ABclonal), anti-GPX4 (1:1,000, C29H4, Cell Signaling Technology), anti-Cox-2 (1:1,000, 9461, Cell Signaling Technology), anti-β-actin (1:2,000, BA2305, Boster), and anti-FTH1 (1:1,000, BA0362, Boster).

Techniques: Negative Control, Produced, Staining, Electron Microscopy, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Age-dependent roles of cardiac remodeling in sepsis defense and pathogenesis

doi: 10.1101/2023.03.14.532695

Figure Lengend Snippet: (A-C) The cardiac transcriptomes of young and old uninfected and LD 50 challenged mice were determined as described in . (A) Venn diagram of the genes induced in young surviving mice compared to all other five conditions are shown; YS – young surviving YU – young uninfected, YD – young dying, OS – old surviving, OU – Old uninfected, OD – old dying. (B) Heat map of the overlap of genes for all five comparisons in (A); (C) KEGG pathway enrichment analysis of the 316 genes that were specifically induced in the young surviving mice compared to the other five conditions shown in (A-B) showing 10 most significant groups. (D) Cardiac gene expression of Foxo1 in uninfected and LD 50 challenged young and old mice when dying mice in each age group reached maximal morbidity (10hrs for young and 24hrs for old). n = 3-5 mice per condition. **** p < 0.0001. Data are also shown in Supplemental Figure 9F. (E) Young mice were infected with a low dose of polymicrobial sepsis and treated with a FoxO1 inhibitor or vehicle and survival was measured. p value is comparing vehicle I to FoxO1 I; n = 5-9 mice per condition. p = 0.0003. (F) Young and old Foxo1 mck cre- and mck° re+ mice were infected with polymicrobial sepsis and survival was monitored. n = 9-11 mice per condition. p = 0.0212 for young and p = 0.0001 for old. (G) Representative heart images from young uninfected and vehicle infected surviving and FoxO1 inhibitor treated dying mice at 10hrs post-infection. Original images are shown in Supplemental Figure 11A. Scale bar – 3mm. (H) Heart weights normalized to body weights from weight-matched young uninfected and infected mice treated with a FoxO1 inhibitor or vehicle at 10hrs post-infection. n = 11-25 mice per condition. *** p < 0.0005, **** p < 0.0001. (I) Values of infected mice from (H) normalized to the average of uninfected values from (H). n = 13-25 mice per condition. **** p < 0.00001. (J) Serum BNP levels of young uninfected, vehicle infected surviving and FoxO1 inhibitor treated dying mice at 10hrs postinfection. n = 10-15 mice per condition. ** p < 0.005, *** p < 0.0005 ****p < 0.0001. (K) Serum Galectin-3 levels of young uninfected, vehicle infected surviving and FoxO1 inhibitor treated dying mice at 10hrs post-infection. n = 10 mice per condition. ** p < 0.01, **** p < 0.0001. (L) Serum Troponin I levels of young uninfected, vehicle infected surviving and FoxO1 inhibitor treated dying mice at 10hrs post-infection. n = 9-18 mice per condition. ** p < 0.005, *** p < 0.0005. Vehicle U – vehicle uninfected, FoxO1 U – FoxO1 inhibitor uninfected, Vehicle I – vehicle infected, FoxO I – FoxO1 inhibitor infected. Error bars +/- SEM. For pairwise comparisons, t-test, One way ANOVA, Kurskal Wallis with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. For survival, Log-rank analysis.

Article Snippet: Samples were quantified with a BCA reaction and subjected to western analysis of GAPDH (Cell Signaling), phospho-FoxO1/FoxO3 (Cell Signaling), FoxO1 plus FoxO3 (Cell Signaling).

Techniques: Expressing, Infection

Journal: bioRxiv

Article Title: Age-dependent roles of cardiac remodeling in sepsis defense and pathogenesis

doi: 10.1101/2023.03.14.532695

Figure Lengend Snippet: (A) Heat map of FoxO1 targets in hearts from young uninfected and LD 50 -challenged mice from RNAseq analysis presented in . n = 3-5 mice per condition. (B-C) 12 and 75 week old mice were infected with the LD 50 dose of polymicrobial sepsis. Hearts were harvested when dying animals in each age group reached maximal morbidity (10 hrs for young and 24 hrs for old) and expression of (B) Trim63 and (C) Fbxo32 were measured. n = 3-5 mice per condition. *** p < 0.0005, **** p < 0.0001. Data are also presented in Supplemental Figure 12C-D. (D-E) Young mice were infected with a low dose (~LD 10 ) of polymicrobial sepsis and treated with a FoxO1 inhibitor or vehicle. Hearts were harvested at 10 hr post-infection and expression of (D) Trim63 and (E) Fbxo32 were measured. n = 8-10 mice per condition. *** p < 0.0005, ****p < 0.0001. (F) Young wild type and Trim63 +/- mice were infected with a low dose of polymicrobial sepsis (~LD25) and survival was determined; n = 5-9 mice per condition. p = 0.0186. (G-L) Wild type and Trim63 +/-,-/- mice were infected with a low dose of polymicrobial sepsis (~LD25). Serum and hearts were harvested ~10 hours post-infection. (G) Representative images of hearts. Scale bar = 3mm. Original images are shown in Supplemental Figure 12H. (H) Weights of hearts normalized to body weight. n = 5-11 mice per condition. * p < 0.05, **p < 0.01, **** p < 0.0001. (I) Infected values from (H) normalized to the average of uninfected values from (H). n = 8-15 mice per condition. *** p = 0.0007. (J) Serum BNP levels. n = 9-13 mice per condition. *** p < 0.0005, **** p < 0.0001. (K) Serum Galectin-3 levels. n = 9-13 mice per condition. ** p <0.005, * p < 0.05. (L) Serum Troponin I levels. n = 3-12 mice per condition. **p < 0.005, *** p < 0.0005. (M) Old infected mice were treated with vehicle or a Trim63 inhibitor and survival was monitored. n = 10 mice per condition. p = 0.0227. (N-Q) 75 week old mice were infected with polymicrobial sepsis and treated with a vehicle or Trim63 inhibitor. Hearts and serum were harvested at 24hrs post-infection. (N) Images of representative hearts harvested from old infected and uninfected mice treated with vehicle or a Trim63 inhibitor. Scale bar = 3mm. Original images are shown in Supplemental Figure 14F. (O) Weights of hearts normalized to body weight. n = 13-23 mice per condition. * p < 0.05, ** p < 0.005, **** p < 0.0001. (P) Infected values from (O) normalized to the average of uninfected values from (O). n = 21-23 mice per condition. **** p < 0.0001. (Q) Serum BNP levels. n = 8-13 mice per condition. ** p < 0.005, *** p < 0.0005. Trim +/+ U = wild type uninfected; Trim63 +/- , -/- U = heterozygous and homozygous Trim63 mutants uninfected; Trim +/+ I = wild type infected; Trim63 +/- -/- I = heterozygous and homozygous Trim63 mutants infected. Vehicle U – vehicle treated uninfected, Trim63 U – Trim63 inhibitor treated uninfected, Vehicle I – vehicle treated infected, Trim63 I – Trim63 inhibitor treated infected Error bars indicate +/- SEM. For pairwise comparisons, t- test, One way ANOVA, Kurskal Wallis with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. For survival, Log-rank analysis.

Article Snippet: Samples were quantified with a BCA reaction and subjected to western analysis of GAPDH (Cell Signaling), phospho-FoxO1/FoxO3 (Cell Signaling), FoxO1 plus FoxO3 (Cell Signaling).

Techniques: Infection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway

doi: 10.3390/ijms24043807

Figure Lengend Snippet: Representative images and quantitative analysis for Akt, FoxOs and eNOS expressions in STZ-induced diabetic mice with or without febuxostat treatment. ( a ) Representative immunoblot showing Akt, FoxOs and eNOS expression levels in mouse kidneys. ( b ) Quantitative analyses for phosphor-Ser 473 Akt/total Akt, ( c ) Quantitative analyses for phospho-Ser 256 FoxO1/total-FoxO1. ( d ) Quantitative analyses for phospho-Ser 253 FoxO3a/total-FoxO3a. ( e ) Quantitative analyses for phospho-Ser 1173 eNOS/total eNOS, * p < 0.05 and *** p < 0.001 vs. Cont, †† p < 0.01 and ††† p < 0.001 vs. Feb and ‡ p < 0.05, ‡‡ p < 0.01, and ‡‡‡ p < 0.001 vs. STZ group.

Article Snippet: The membrane was incubated with primary antibodies against the following target proteins: VEGFR1 (Boster, Pleasanton, CA, USA), VEGFR2 (St John’s Laboratory, London, UK), VEGFR3 (NSJ Bioreagent Logo, San Diego, CA, USA), NOX1 and NOX4 (Lifespan Biosciences, Seattle, WA, USA), NOX2 (BD Biosciences, San Jose, CA, USA), total Akt, phospho-Ser 473 Akt, SOD1 and SOD2 (Cell Signaling Technology, Danvers, MA, USA), total FoxO1, phospho-Ser 256 FoxO1, total FoxO3a, and phospho-Ser 253 FoxO3a (Novus Biologicals, Centennial, CO, USA), total endothelial nitric oxide synthase (eNOS, EMD Millipore, Middlesex County, MA, USA), and phospho-Ser 1173 eNOS (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing

Journal: Frontiers in Endocrinology

Article Title: Characterization of placental endocrine function and fetal brain development in a mouse model of small for gestational age

doi: 10.3389/fendo.2023.1116770

Figure Lengend Snippet: List of antibodies used for western blotting.

Article Snippet: Phospho-FOXO1 (Ser256) , Cell Signalling, 9461 , 1/1,000.

Techniques: Western Blot

Journal: The Journal of Experimental Medicine

Article Title: PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A–producing γδ T cells

doi: 10.1084/jem.20211431

Figure Lengend Snippet: PD-1 signaling regulates expansion of lung Vγ6 + cells. (A) CD3 + T cells were isolated from the lungs of FVB/n mice and stimulated with recombinant IL-1β and IL-23 in the presence of plate-bound PD-L1-Fc or plate-bound anti-ICOS for 24 h. Supernatants were examined for IL-17A levels by ELISA. Each dot represents cells from one mouse ( n = 5–7/group). Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s posthoc test; **P < 0.01, ***P < 0.001. (B) WT FVB/n mice were injected with a single dose of 200 μg anti–PD-1 or anti-ICOS followed by injections of 100 μg for two consecutive days. Control mice followed the same dosage regime with isotype control. Mice were sacrificed 24 h after the third injection. Single-cell suspensions from lung were stimulated for 3 h with PMA, ionomycin, and Brefeldin A. Cells were stained with antibodies against CD3, TCRδ, CD27, Vγ4, Vγ6, and IL-17A. The proportion of cells expressing IL-17A, the MFI of IL-17A, and the absolute number of cells is represented graphically for both lung Vγ4 + and Vγ6 + CD27 − cells. Each dot represents one mouse. Data are presented as mean ± SD ( n = 6–7 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01. (C) Percentage and total numbers of neutrophils in isotype control, anti–PD-1- and anti-ICOS–treated mice as measured by IDEXX ProCyte hematology analyzer. Each dot represents one mouse. Data are presented as mean ± SD ( n = 6–7 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01. (D) Representative Western blot analysis (from three biological replicates) of phospho-FOXO1 and total FOXO1 levels in γδ T cells from lungs of FVB/n mice stimulated as depicted. (E) CD3 + T cells were isolated from the lungs of FVB/n mice and cultured with plate-bound PD-L1-Fc for 3 h. Cells were stimulated with recombinant IL-1β and IL-23 for the last 30 min. Cells were analyzed by flow cytometry. Representative histograms are shown, and combined data are represented graphically. Each dot represents one mouse. Data are presented as mean ± SD ( n = 4 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01.

Article Snippet: Phospho-FOXO1 , N/A , , Cell Signaling , 9464 , 1:50.

Techniques: Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Injection, Staining, Expressing, Western Blot, Cell Culture, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A–producing γδ T cells

doi: 10.1084/jem.20211431

Figure Lengend Snippet: Antibodies utilized for flow cytometry

Article Snippet: Phospho-FOXO1 , N/A , , Cell Signaling , 9464 , 1:50.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A–producing γδ T cells

doi: 10.1084/jem.20211431

Figure Lengend Snippet: PD-1 signaling regulates expansion of lung Vγ6 + cells. (A) CD3 + T cells were isolated from the lungs of FVB/n mice and stimulated with recombinant IL-1β and IL-23 in the presence of plate-bound PD-L1-Fc or plate-bound anti-ICOS for 24 h. Supernatants were examined for IL-17A levels by ELISA. Each dot represents cells from one mouse ( n = 5–7/group). Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s posthoc test; **P < 0.01, ***P < 0.001. (B) WT FVB/n mice were injected with a single dose of 200 μg anti–PD-1 or anti-ICOS followed by injections of 100 μg for two consecutive days. Control mice followed the same dosage regime with isotype control. Mice were sacrificed 24 h after the third injection. Single-cell suspensions from lung were stimulated for 3 h with PMA, ionomycin, and Brefeldin A. Cells were stained with antibodies against CD3, TCRδ, CD27, Vγ4, Vγ6, and IL-17A. The proportion of cells expressing IL-17A, the MFI of IL-17A, and the absolute number of cells is represented graphically for both lung Vγ4 + and Vγ6 + CD27 − cells. Each dot represents one mouse. Data are presented as mean ± SD ( n = 6–7 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01. (C) Percentage and total numbers of neutrophils in isotype control, anti–PD-1- and anti-ICOS–treated mice as measured by IDEXX ProCyte hematology analyzer. Each dot represents one mouse. Data are presented as mean ± SD ( n = 6–7 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01. (D) Representative Western blot analysis (from three biological replicates) of phospho-FOXO1 and total FOXO1 levels in γδ T cells from lungs of FVB/n mice stimulated as depicted. (E) CD3 + T cells were isolated from the lungs of FVB/n mice and cultured with plate-bound PD-L1-Fc for 3 h. Cells were stimulated with recombinant IL-1β and IL-23 for the last 30 min. Cells were analyzed by flow cytometry. Representative histograms are shown, and combined data are represented graphically. Each dot represents one mouse. Data are presented as mean ± SD ( n = 4 mice/group). One-way ANOVA followed by Dunnett’s posthoc test; **P < 0.01.

Article Snippet: Antibodies not previously described include anti-phospho-STAT3 (1:2,000; #9145; Cell Signaling Technology), anti-STAT3 (1:1,000; #9139; Cell Signaling Technology), anti-phospho-p65 (1:1,000; #3033; Cell Signaling Technology), anti-p65 (1:1,000; #8242; Cell Signaling Technology), anti-phospho-FOXO1/FOXO3A (1:1,000; #9464; Cell Signaling Technology), and anti-FOXO1 (1:1,000; #18592-1-AP; Proteintech).

Techniques: Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Injection, Staining, Expressing, Western Blot, Cell Culture, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: PD-1 and TIM-3 differentially regulate subsets of mouse IL-17A–producing γδ T cells

doi: 10.1084/jem.20211431

Figure Lengend Snippet: Antibodies utilized for flow cytometry

Article Snippet: Antibodies not previously described include anti-phospho-STAT3 (1:2,000; #9145; Cell Signaling Technology), anti-STAT3 (1:1,000; #9139; Cell Signaling Technology), anti-phospho-p65 (1:1,000; #3033; Cell Signaling Technology), anti-p65 (1:1,000; #8242; Cell Signaling Technology), anti-phospho-FOXO1/FOXO3A (1:1,000; #9464; Cell Signaling Technology), and anti-FOXO1 (1:1,000; #18592-1-AP; Proteintech).

Techniques:

Journal: Cell Death & Disease

Article Title: SGK2 promotes prostate cancer metastasis by inhibiting ferroptosis via upregulating GPX4

doi: 10.1038/s41419-023-05614-5

Figure Lengend Snippet: A , B Levels of cytoplasmic and nuclear FOXO1/FOXO4 protein in PCa cells with SGK2 overexpression were determined by western blotting. α-Tubulin and Histone-H3 were used as cytoplasmic and nuclear markers, respectively. C Location of FOXO1/FOXO4 in PCa cells with SGK2 overexpression was detected by immunofluorescence staining. Scale bars, 10 μm. D Position weight matrix of FOXO1 binding site motif from TRANSFAC database. E Pattern of four predicted transcription factor binding sites of FOXO1 and GPX4 promoter by using the ConTra V2 database, and corresponding mutant sites. (TFBS, transcription factor binding sites). F The luciferase activity of wild-type (wt) GPX4 promoter after transfection with FOXO1/FOXO4 overexpressed plasmids in PC3 cells. G The luciferase activities of the four mutant transcription factor binding sites of GPX4 promoter after transfection with FOXO1 overexpressed plasmids in PC3 cells. H The luciferase activity of GPX4 promoter after transfection with SGK2 overexpressed plasmids and FOXO1 overexpressed plasmids independently or in combination in PC3 cells. I , J Western blot assay showed the levels of GPX4 protein in the indicated group. Data are presented as representative images or as the mean ± SD of three independent experiments. * P < 0.05; ns, not significant.

Article Snippet: The following primary antibodies were used: SGK2 (5595, Cell Signaling Technology), GPX4 (67763-1-Ig, Proteintech), SLC3A2 (15193-1-AP, Proteintech), AIFM2 (20886-1-AP, Proteintech), SLC7A11 (12691, Cell Signaling Technology), ACSL4 (22401-1-AP, Proteintech), DHODH (14877-1-AP, Proteintech), GAPDH (10494-1-AP, Proteintech), α-Tubulin (66031-1-Ig, Proteintech), Histone-H3 (17168-1-AP, Proteintech), FOXO1 (66457-1-Ig, Proteintech), FOXO3 (10849-1-AP, Proteintech), FOXO4 (21535-1-AP, Proteintech), FOXO6 (19122-1-AP, Proteintech), Phospho-FOXO1 (Ser-256) (ab131339, Abcam), Phospho-FOXO1 (Ser-319) (ab47326, Abcam), and Phospho-FOXO1 (Thr-24) (9464, Cell Signaling Technology).

Techniques: Over Expression, Western Blot, Immunofluorescence, Staining, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection

Journal: Cell Death & Disease

Article Title: SGK2 promotes prostate cancer metastasis by inhibiting ferroptosis via upregulating GPX4

doi: 10.1038/s41419-023-05614-5

Figure Lengend Snippet: A Demonstrative immunofluorescence images of SGK2 and FOXO1 protein location in PC3 and DU145 cells. Scale bars, 10 μm. B , C Cross-linking of SGK2 and FOXO1 in PCa cells with SGK2 overexpression was detected by co-immunoprecipitation experiment with anti-SGK2 antibody and anti-FOXO1 antibody. The lysate immunoprecipitated with anti-immunoglobulin G antibody was served as negative control. D , E Western blotting analysis of the expression level of FOXO1 phosphorylation at Thr-24(T24) and Ser-319(S319) in PCa cells stably overexpressing SGK2. F On the basis of SGK2 overexpression, levels of cytoplasmic and nuclear FOXO1 protein in the indicated group by western blotting. α-Tubulin and Histone-H3 were used as cytoplasmic and nuclear markers, respectively. G On the basis of SGK2 overexpression, western blotting analysis of the expression level of GPX4 in PCa cells transfected with FOXO1 overexpressed plasmids of wt, T24A, S319A, Both, respectively. H , I Cell viability was detected by CCK-8 assay in the indicated group. J FOXO1 and GPX4 expression levels were assessed using western blotting in PCa cells with/without SGK2 overexpression. F – J wt, wild-type FOXO1 overexpressed plasmids; T24A, mutant T24A FOXO1 overexpressed plasmids; S319A, mutant S319A FOXO1 overexpressed plasmids; Both, mutant T24A and S319A FOXO1 overexpressed plasmids. Data are presented as representative images or as the mean ± SD of three independent experiments. * P < 0.05; ns, not significant.

Article Snippet: The following primary antibodies were used: SGK2 (5595, Cell Signaling Technology), GPX4 (67763-1-Ig, Proteintech), SLC3A2 (15193-1-AP, Proteintech), AIFM2 (20886-1-AP, Proteintech), SLC7A11 (12691, Cell Signaling Technology), ACSL4 (22401-1-AP, Proteintech), DHODH (14877-1-AP, Proteintech), GAPDH (10494-1-AP, Proteintech), α-Tubulin (66031-1-Ig, Proteintech), Histone-H3 (17168-1-AP, Proteintech), FOXO1 (66457-1-Ig, Proteintech), FOXO3 (10849-1-AP, Proteintech), FOXO4 (21535-1-AP, Proteintech), FOXO6 (19122-1-AP, Proteintech), Phospho-FOXO1 (Ser-256) (ab131339, Abcam), Phospho-FOXO1 (Ser-319) (ab47326, Abcam), and Phospho-FOXO1 (Thr-24) (9464, Cell Signaling Technology).

Techniques: Immunofluorescence, Over Expression, Immunoprecipitation, Negative Control, Western Blot, Expressing, Stable Transfection, Transfection, CCK-8 Assay, Mutagenesis

Journal: Cell Death & Disease

Article Title: SGK2 promotes prostate cancer metastasis by inhibiting ferroptosis via upregulating GPX4

doi: 10.1038/s41419-023-05614-5

Figure Lengend Snippet: The upregulation of SGK2 increased the nuclear exclusion of FOXO1 via phosphorylating FOXO1 and indirectly upregulated GPX4 expression, which ultimately suppressed ferroptosis and promoted PCa metastasis.

Article Snippet: The following primary antibodies were used: SGK2 (5595, Cell Signaling Technology), GPX4 (67763-1-Ig, Proteintech), SLC3A2 (15193-1-AP, Proteintech), AIFM2 (20886-1-AP, Proteintech), SLC7A11 (12691, Cell Signaling Technology), ACSL4 (22401-1-AP, Proteintech), DHODH (14877-1-AP, Proteintech), GAPDH (10494-1-AP, Proteintech), α-Tubulin (66031-1-Ig, Proteintech), Histone-H3 (17168-1-AP, Proteintech), FOXO1 (66457-1-Ig, Proteintech), FOXO3 (10849-1-AP, Proteintech), FOXO4 (21535-1-AP, Proteintech), FOXO6 (19122-1-AP, Proteintech), Phospho-FOXO1 (Ser-256) (ab131339, Abcam), Phospho-FOXO1 (Ser-319) (ab47326, Abcam), and Phospho-FOXO1 (Thr-24) (9464, Cell Signaling Technology).

Techniques: Expressing

Journal: Molecular Medicine

Article Title: TOP2A deficiency leads to human recurrent spontaneous abortion and growth retardation of mouse pre-implantation embryos

doi: 10.1186/s10020-022-00592-4

Figure Lengend Snippet: Downregulation of TOP2A affects the FOXO signaling pathway. A Western blotting showed changes in the levels of phosphorylated- FoxO1, phosphorylated-FoxO3a and phosphorylated-Rb after TOP2A downregulation. B Western blotting was used to assess changes in the level of phosphorylated-FoxO1 in the inhibitor-treated group. C q-PCR assay was used to verify changes in the mRNA expression of several downstream signaling molecules. D mRNA expression of cycle-associated genes was assessed after TOP2A knock down in JEG-3 cells and after treatment with chemical inhibitors that reduce FoxO1 activity. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Equal amounts of protein (30 μg/lane) were separated using SDS–PAGE and transferred on to PVDF membranes (Millipore Sigma, Burlington, MA, USA), which was subsequently blocked with quick blocking buffer (NCM Biotech, Suzhou, China) for 15 min. Membranes were washed with phosphate-buffered saline (PBS) and then incubated with the following primary antibodies overnight at 4 °C: TOP2A (1:10,000; Abcam, Cambridge, UK), matrix metalloproteinase 9 (MMP9) (1:1000; Abcam), MMP2 (1:1000; Abcam), cyclin-dependent kinase 2 (CDK2) (1:1000, Cell Signaling Technology, Danvers, MA, USA), CDK4 (1:1000, Cell Signaling Technology), phosphor-FoxO1 (1:1000, Cell Signaling Technology), P21 (1:1000; Cell Signaling Technology), P27 (1:1000; Cell Signaling Technology), cleaved caspase 3 (1:1000; Abcam), caspase 3 (1:1000; Abcam), and β-actin (1:2000; Proteintech, Wuhan, China).

Techniques: Western Blot, Expressing, Activity Assay