Journal: The Journal of Physiology

Article Title: Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5

doi: 10.1113/jphysiol.2012.240952

Figure Lengend Snippet: Representative immunofluorescence images of PLIN5 (A), IMTG (B), and the merged images (C) with the yellow areas describing regions of overlap between PLIN5 and IMTG images. The yellow objects were then extracted (to measure the number of PLIN5-LDs; D), and the number subtracted from the total number of LDs to quantify the number of PLIN5-null-LD. The same procedure was used to obtain values of colocalization between PLIN2 and IMTG. White bar, 5 μm. The number of PLIN2-LD (filled bars) and PLIN2-null-LD (open bars) were quantified before (PreEx) and after exercise (PostEx), prior to (E) and following (F) training in both the SIT and ET groups. The same analysis was repeated for PLIN5 for pre- (G) and post-training (H). *Main effect of exercise bout (P < 0.05 vs. pre-exercise).

Article Snippet: The specificity of the PLIN5 antibody was confirmed in a competition experiment in which a PLIN5 recombinant peptide (Cat. no. NB110–60511PEP; Novus Biologicals, Cambridge, UK) was incubated with the PLIN5 primary antibody, subsequently resulting in removal of the positive fluorescent signal for PLIN5 (Supplementary Fig. S1 ).

Techniques: Immunofluorescence

Journal: The Journal of Physiology

Article Title: Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5

doi: 10.1113/jphysiol.2012.240952

Figure Lengend Snippet: Representative immunofluorescence images of PLIN5 (stained green) in combination with WGA to identify the cell border (stained blue) in skeletal muscle, pre- (A) and post- (B) 6 weeks of SIT. Type I fibres are indicated with a ‘I’, all other fibres are assumed type II fibres. White bar, 50 μm. PLIN5 expression, quantified from immunofluorescence images of PLIN5, in type I and type II fibres obtained before (C) and after (D) 6 weeks of SIT (filled bars) or ET (open bars). PLIN5 expression quantified from immunofluorescence images correlated with PLIN5 expression determined following immunoblotting of whole muscle homogenates (E). Values are presented as means ± SEM (n= 8 per group). *Main effect of fibre type (P < 0.05 vs. type I fibres). †Main effect of training intervention (P < 0.05 vs. pre-training).

Article Snippet: The specificity of the PLIN5 antibody was confirmed in a competition experiment in which a PLIN5 recombinant peptide (Cat. no. NB110–60511PEP; Novus Biologicals, Cambridge, UK) was incubated with the PLIN5 primary antibody, subsequently resulting in removal of the positive fluorescent signal for PLIN5 (Supplementary Fig. S1 ).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot

Journal: The Journal of Physiology

Article Title: Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5

doi: 10.1113/jphysiol.2012.240952

Figure Lengend Snippet: Bivariate correlation analysis

Article Snippet: The specificity of the PLIN5 antibody was confirmed in a competition experiment in which a PLIN5 recombinant peptide (Cat. no. NB110–60511PEP; Novus Biologicals, Cambridge, UK) was incubated with the PLIN5 primary antibody, subsequently resulting in removal of the positive fluorescent signal for PLIN5 (Supplementary Fig. S1 ).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: List of antibodies and antibody-blocking peptides used in the study.

Article Snippet: BAK antibody blocking peptide , ELISA detection , N/A , N/A , Novus Biologicals , NBP1-77152PEP , N/A.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Peptide ELISA, Activity Assay

Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 3. Decreased glutathione pools by CHAC1 reduces protein-SSG and aggravates APAP-induced hepatotoxicity and ferroptosis in APAP-injured mice liver. (A) Quantification of GSH in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (B) Quantification of GSSG in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad- CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (C) Western blot analysis of S-glu tathionylated proteins in the liver tissues of Chac1+/+ Ad-GFP and Chac1−/−Ad-GFP mice treated with 300 mg/kg APAP for 2 and 6 h. GAPDH was used as an internal reference, followed by quantification of relative levels of S-glutathionylated proteins after 6 h of APAP treatment (Data are mean ± SEM of n = 3 mice/ group, t-test). (D) Western blot analysis of S-glutathionylated proteins and CHAC1-FLAG protein in the liver tissues of Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. GAPDH was used as an internal reference. Statistical chart shows the relative expression levels of S-glutathionylated proteins 6 h after APAP treatment (Data are mean ± SEM of n = 4 mice/group, t-test). (E) Serum levels of ALT and AST in Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h (Data are mean ± SEM of n = 5 mice/group, t-test). (F) H&E staining, 4-HNE protein adduct staining in liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. Scale bars = 200 μm. The area of liver injury and immunohistochemical score for 4-HNE protein adduct staining were quantified (Data are mean ± SEM of n = 5 mice/group, t-test). APAP, acetaminophen; GSH, glutathione; GSSG, oxidized glutathione; ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, haematoxylin and eosin; Chac1−/−, Chac1- deficient; Chac1+/+, wild-type controls.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Saline, Western Blot, Expressing, Staining, Immunohistochemical staining

Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 4. Redox proteomic analysis reveals that the S-glutathionylation of Cys90 on ARF6 is regulated by CHAC1 in ferroptotic PMHs induced by APAP. (A) Flowchart outlining the key experimental procedures for proteomic analysis of S-glutathionylation. (B) Scatter plot illustrating the distribution of differential modification sites, sorted by the ratio of Ad-GFP + APAP/Ad-GFP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences, and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (C) Scatter plot showing the distribution of differential modification sites, sorted by the ratio of Ad-CHAC1 + APAP/Ad-GFP + APAP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (D) Venn diagram showing differentially modified sites under both APAP stimulation and CHAC1 overexpression (Fold change ≥1.2). (E) The heat map illustrating the union of differential modification sites in Ad-GFP, Ad-GFP + APAP, Ad-CHAC1, and Ad-CHAC1 + APAP comparison groups (CV < 0.1, fold change ≥1.2). (F) Scatter plot showing differentially modified sites under both APAP stimulation and CHAC1 overexpression; the order was sorted by the ratio of Ad-GFP + APAP/Ad-GFP (CV < 0.1, fold change ≥1.2). (G) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in PMHs. The secondary mass spectrum shows fragment ion information of the ARF6 C90 peptide segment. (H) Histogram showing the relative modification abundance of ARF6 C90 in different treatment groups, with glutathionylated peptides identified and quantified by LC-MS/MS (All values were normalized by the mean of the AdGFP-CON group, data are mean ± SEM of n = 2 biologically independent samples). (I) IP assay showing the expression of S-glutathionylated ARF6 in 293T cells overexpressing Myc-tagged ARF6. Whole cell lysates were used to confirm the expression of ARF6. (J) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in 293T cells overexpressing Myc-tagged ARF6. The secondary mass spectrum shows fragment ion infor mation of the ARF6 C90 peptide segment. PMH, primary mouse hepatocyte; IP, immunoprecipitation.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Modification, Over Expression, Comparison, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Immunoprecipitation

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A,B) Mice were injected with 0.25×105 (n=5, tumor onset in 4 of 5 injected mice), 0.5×105 (n=5), 1×105 (n=5, one mouse died before FACS analysis), 2×105 B16 cells (n=5, one mouse died before FACS analysis), and 2 weeks later 4PD1hi and Tregs (percentage of total CD4+) were analyzed in spleen (SP), tumor-draining lymph nodes (DLNs) and tumor (TM) in comparison with spleens from naive mice (SP naive) (mean ± SEM; unpaired t test) (A). Pearson correlation analyses of tumor burden and intra-tumor 4PD1hi %, Tregs % and the indicated intra-tumor T-cell ratios (B). (C) Percentage of 4PD1hi among CD4+ T cells in healthy donors’ PB (n=7), advanced melanoma patients’ PB (n=47) and tumors (TM, n=10); NSCLC patients’ PB (n=51) and tumors (TM, n=10) (mean ± SEM; unpaired t test), and representative plots of Foxp3 and PD-1 expression in CD4+CD45+ T cells, and CD25 expression in 4PD1hi, Tregs and 4PD1− from the indicated samples. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S1.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Injection, Expressing

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) Schema of in vitro suppression assay of CD45.1+ T cells (target) with 4PD1hi, 4PD1− or PD-1−Tregs FACS-sorted from spleens of naive Foxp3-GFP mice (CD45.1−, effectors). (B) CTV dilution and frequency of CD44+CD25+ in total CD45.1+CD4+ target T cells in the indicated co-cultures at the indicated effector:target ratios after 48-hr incubation (mean ± SD; n=2; 2-way ANOVA, 4PD1hi and Tregs vs 4PD1−). (C) Quantification of IFN-γ, TNF-α and IL-2 by FACS-based bead immunoassay in culture supernatants of CD4+ cells alone or co-cultured with the indicated cells for 48 hr (ratio 1:1; mean ± SD; n=2; unpaired t test). (D) Foxp3, CD25 and PD-1 MFI in effector CD45.1−CD4+ T-cell subsets co-cultured with CD45.1+CD4+ target T cells (ratio 1:1) for 48 hr (mean ± SD; n=2; unpaired t test). (E) In vivo suppression assay with 4PD1hi or Tregs FACS-sorted from B16-bearing Foxp3-GFP mice and co-transferred with CFSE-labeled Pmel/gp100-TCR-specific CD8+ T cells (Pmels) (1:1 ratio) into irradiated CD45.1+ recipients and stimulated in vivo with irradiated B16 cells (schema). Proliferation (CFSE dilution) and activation (CD44 and CD25 expression) of CD45.1−Thy1.1+CD8+ Pmels in recipient spleens (mean ± SEM; n=4–5; unpaired t test). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S2.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: In Vitro, Suppression Assay, Incubation, Cell Culture, In Vivo, Labeling, Irradiation, Activation Assay, Expressing

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) FACS gating strategy to sort human 4PD1hi, 4PD1− and Tregs based on PD-1 and CD25 expression in CD4+ T cells and related Foxp3 expression (left). Proliferation (CTVlow) and activation (CD25 MFI) of autologous target CD4+ T cells co-cultured with donor-derived 4PD1hi, 4PD1− and Tregs at 1:1 ratio for 72 hr (middle) (mean ± SD; n=3; unpaired t test, 4PD1hi and Tregs vs 4PD1−). Heatmap with unsupervised hierarchical clustering of the indicated cytokines in co-culture supernatants as assessed by Luminex-based bead immunoassay (right). (B,C) Proliferation (CTVlow) of target autologous (auto) CD4+ TILs (B) or donor-derived allogeneic circulating CD8+ T cells (C) co-cultured with human tumor-infiltrating 4PD1hi, 4PD1− or Tregs (1:1 ratio) for 72 (B) or 96 hr (C), and cytokine production by Luminex-based bead immunoassay in the same cultures. Mean ± SD (B melanoma, n=2 with Tregs and 4PD1hi, n=6 with 4PD1−; B NSCLC, n=2 with 4PD1hi, n=3 with 4PD1− and Tregs; C, n=3); unpaired t test, 4PD1hi and Tregs vs 4PD1−. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S3.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Expressing, Activation Assay, Cell Culture, Derivative Assay, Co-Culture Assay, Luminex

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) Fold changes in circulating 4PD1hi (percentage of total CD4+) in advanced NSCLC patients during treatment with nivo3 (nivolumab 3 mg/kg, once every 2 weeks (q2wks), n=10), nivo3+ipi1 (nivolumab 3 mg/kg + ipilimumab 1 mg/kg, q3wks, q6wks+q2wks, or q12wks+q2wks, n=21), nivo1+ipi1 (nivolumab 1 mg/kg + ipilimumab 1mg/kg, q3wks, or q6wks, n=11) or nivo1+ipi3 (nivolumab 1mg/kg + ipilimumab 3mg/kg, q3wks, n=8) (average ± SEM; 2-way ANOVA with Bonferroni correction, nivo3 vs nivo1+ipi1 and nivo3 vs nivo1+ipi3). Representative FACS plots of Foxp3 and PD-1 expression in CD4+ T cells from NSCLC patients treated with nivo1+ipi3 (red) or nivo3 (blue) at the indicated time points. (B) Circulating 4PD1hi (percentage of CD4+) in B16-bearing mice treated with αCTLA-4 monotherapy (100 μg or 300 μg/cycle; average ± SEM, n=7–10) relative to naive mice (n=5) (2-way ANOVA with Bonferroni correction, treated vs naive mice). (C) Pairwise comparison of 4PD1hi (percentage of CD4+) at the indicated time points relative to baseline in advanced melanoma patients during ipilimumab (ipi, 3 mg/kg, q3wks; n=47) or pembrolizumab treatment (pembro, 2 or 10 mg/kg, q3wks; n=52) (average ± SEM) (left). Representative FACS plots of Foxp3 and PD-1 expression in CD4+ T cells from melanoma patients treated with ipi (red) or pembro (blue) at the indicated time points (middle). Pairwise comparison of circulating 4PD1hi/CD4+ % at baseline and 3 weeks after pembro (right). (D) Average ± SEM tumor diameter (left; n=10; 2-way ANOVA with Bonferroni correction) and Kaplan-Meier tumor-free survival curves (right; pooled data from 3 independent experiments, n=30; log-rank test; number of tumor-free mice approximately 100 days after tumor implantation is reported for each group) of B16-bearing mice treated with VRP-TRP2 and αCTLA-4 and/or αPD-1 as indicated. (E) Intra-tumor 4PD1hi and Tregs frequencies one day after treatment completion in B16-bearing mice treated as in D (average ± SEM; n=9–10; unpaired t test). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S4.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Expressing, Tumor Implantation

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) In vitro suppression assays with 4PD1hi, Tregs and 4PD1− from spleens of sRBC-immunized or control B16-bearing Foxp3-GFP mice at the indicated ratios. Mean ± SD proliferation (CTVlow) and activation (CD25+CD44+) of target CD4+ T cells, and Foxp3 and PD-1 MFI in CD45.1− 4PD1hi, 4PD1− or Tregs from the same co-cultures after 48-hr incubation (n=2–3; 2-way ANOVA, NT-4PD1hi vs sRBC-4PD1hi). (B) In vitro suppression assays with CXCR5+ and CXCR5− 4PD1hi, 4PD1− and Tregs from spleens (SP) and tumors (TM) of naive and B16-bearing (TB) Foxp3-GFP mice immunized with sRBC. Mean ± SD proliferation (CTVlow) of target CD45.1+CD4+ T cells and quantification of IL-2 by Luminex-based bead immunoassay in culture supernatants after 72-hr incubation with effector cells (1:1 ratio; 4×104 cells from SP; 1×104 cells from TM; n=2–4; unpaired t test). (C) In vitro suppression assays with 4PD1hi, CD44hiPD-1−Foxp3−CD4+ Tmem and Foxp3+ Tregs from spleens of sRBC-immunized Foxp3-GFP mice at the indicated effector:target ratios. Mean ± SD proliferation (CTVlow) and activation (CD25+CD44+) of target CD8+ and CD4+ T cells from 48- and 72-hr co-cultures respectively (n=2–3; 2-way ANOVA, 4PD1hi and Tregs vs Tmem). (D) Average ± SEM tumor diameter of B16-bearing Sh2d1a (SAP) KO and WT C57BL/6J mice treated with αCTLA-4 or control isotype IgG (100 μg x4) starting on day 7 after tumor implantation (suboptimal treatment) (n=4–5; 2-way ANOVA with Bonferroni correction). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S7.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: In Vitro, Activation Assay, Incubation, Luminex, Tumor Implantation

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Purification, Blocking Assay, Recombinant, Staining, Expressing, Knock-Out, Transgenic Assay, Software