Journal: Cell & Bioscience

Article Title: Overexpression of ONECUT1 suppresses hepatoblastoma progression via modulating tumor cell growth and tumor microenvironment

doi: 10.1186/s13578-026-01569-0

Figure Lengend Snippet: Depletion of T cells partially inhibits ONECUT1-induced hepatoblastoma regression in YAPS127A/ΔN90-β-catenin mice. A Experimental design. FVB/N mice were hydrodynamically injected with YAPS127A/ΔN90-β-catenin/TRE-ONECUT1 plasmids on Day − 24. On Day − 5, mice received i.p. injections of control IgG or anti-CD4 plus anti-CD8 antibodies (CD4/CD8-Ab). On Day 0, DOX treatment was initiated to induce ONECUT1 expression or regular water as a DOX (−) control, accompanied by the same antibody regimen. Mice were sacrificed on Day 3 after DOX induction or the corresponding time point in DOX (−) groups. Four experimental groups were included: DOX (+) + IgG, DOX (+) + CD4/CD8-Ab, DOX (−) + IgG, and DOX (−) + CD4/CD8-Ab. B , C Quantification of liver weight ( B ) and liver-to-body weight ratio ( C ). Under DOX (+) conditions, T-cell depletion significantly attenuated ONECUT1-induced tumor regression (* P < 0.05). In contrast, under DOX (−) conditions, CD4/CD8 depletion did not significantly affect liver weight or liver-to-body weight ratio. Statistical significance was determined by a two-tailed unpaired Student’s t-test. Data are presented as mean ± SD. ns, not significant; * P < 0.05; *** P < 0.001; **** P < 0.0001. D Representative gross liver morphology and histological analyses. Images from DOX (+) groups are shown. H&E staining (40×) shows larger tumor burden in CD4/CD8-depleted mice. Ki67 immunostaining (200×) indicates higher proliferation in CD4/CD8-Ab–treated livers. CD4 and CD8 immunostaining (200×) confirms successful T-cell depletion.

Article Snippet: To deplete CD4+ and CD8+ T cells in mice, anti-mouse CD4 monoclonal antibody (clone GK1.5, Purified in vivo GOLDTM Functional Grade, Leinco Technologies) and anti-mouse CD8 monoclonal antibody (clone YTS 169, Purified in vivo GOLDTM Functional Grade, Leinco Technologies) were administered.

Techniques: Injection, Control, Expressing, Two Tailed Test, Staining, Immunostaining

Journal: bioRxiv

Article Title: Systemic Cysteine Elevation Sustains T-Cell Activation to Potentiate PD-1 Blockade

doi: 10.64898/2026.03.16.711688

Figure Lengend Snippet: (A-E) Tumor-infiltrated CD45 + (A), CD4 + (B), CD8 + (C), activated CD4 + (D), and CD8 + (E) (define as CD25 or/and CD69 positive) T cells detected by flow cytometry from KPC tumor-bearing mice receiving isotype IgG (n=11) or anti-PD-1 injections combined with ddH 2 O (n=11) or probiotics (n=8) gavage for 6 weeks. (F)) Representative original immunofluorescence and HALO analysis images of tumor sections staining with CD4 (in green), Ly-6G (in yellow), and FOXP3 (in red). (G and H) Tumor-infiltrated CD4 + (G) and Ly-6G + (H) cells quantified by HALO platform (n=5 per group). (I) Schematic of treatments in KC transgenic PDAC mice. Created with BioRender.com. (J) Tumor weight of KC transgenic mice receiving isotype IgG (n=5) or anti-PD-1 injections combined with ddH 2 O (n=7) or probiotics (n=13) gavage for 6 weeks. (K-O) Tumor-infiltrated CD45 + (K), CD4 + (L), CD8 + (M), activated CD4 + (N), and CD8 + (O) (defined as CD25 or/and CD69 positive) T cells detected by flow cytometry from KC mice receiving isotype IgG (n=3) or anti-PD-1 injections combined with ddH 2 O (n=7) or probiotics (n=3) gavage for 6 weeks. Data are represented as mean ± SEM, whereas ns p>0.05, *p<0.05, **p<0.01, and ****p<0.0001 by one-way ANOVA followed by Fisher’s LSD tests. See also Figure S2.

Article Snippet: Sections were then incubated with CD8 alpha (D4W2Z) Rabbit Monoclonal Antibody (Cell Signaling Technology, 98941) at a 1:400 dilution for 30 minutes at room temperature.

Techniques: Flow Cytometry, Probiotics, Immunofluorescence, Staining, Transgenic Assay

Journal: bioRxiv

Article Title: Systemic Cysteine Elevation Sustains T-Cell Activation to Potentiate PD-1 Blockade

doi: 10.64898/2026.03.16.711688

Figure Lengend Snippet: (A) Line plot connecting group-mean delta copies per million (CPM) for cysteine-related pathways between anti-PD-1(n=8) and combination(n=13) groups. Statistical significance is indicated by thicker line weights and asterisks, whereas *p<0.05. (B) Relative serum cysteine levels of the KPC tumor-bearing mice receiving anti-PD-1 (n=15) or combination (n=10) treatment for 6 weeks. (C) Correlation between serum cysteine levels and tumor weights (n=13). The Pearson correlation coefficient was used to assess the strength and direction of the relationship. (D) Tumor tissue cysteine levels of the KPC tumor-bearing mice receiving anti-PD-1 (n=5) or combination (n=4) treatment for 6 weeks. (E) Correlation between KPC tumor tissue cysteine levels and tumor weights (n=9). The Pearson correlation coefficient was used to assess the strength and direction of the relationship. (F and G) The number of activated CD4 + (F) and CD8 + (G) (defined as CD25 or/and CD69 positive) T cells in mesenteric lymph nodes were detected by flow cytometry from KPC tumor-bearing mice receiving anti-PD-1 (n=12) or combination (n=6) treatment for 6 weeks. Data are represented as mean ± SEM, whereas ns p>0.05 and **p<0.01 by unpaired two-tailed Student’s t-tests (B, D, F, and G). See also Table S2.

Article Snippet: Sections were then incubated with CD8 alpha (D4W2Z) Rabbit Monoclonal Antibody (Cell Signaling Technology, 98941) at a 1:400 dilution for 30 minutes at room temperature.

Techniques: Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Systemic Cysteine Elevation Sustains T-Cell Activation to Potentiate PD-1 Blockade

doi: 10.64898/2026.03.16.711688

Figure Lengend Snippet: Murine splenic T cell growth curve. (B and C) Absolute number per well (B) and viability (C) of murine splenic unactivated T cells after 3 days of culturing. (D) Murine splenic T cell growth curve with anti-CD3 and anti-CD28 activation. (E and F) Absolute number per well (E) and viability (F) of murine splenic T cells following 6 days of anti-CD3 and anti-CD28 activation. (G) Representative T-distributed Stochastic Neighbor Embedding (t-SNE) plots of 6 days-activated murine splenic T cells. (H-K) The proportions of activated subsets (defined as CD25 or/and CD69 positive) (H and I) and central memory subsets (defined as CD44 and CD69 double-positive) (J and K) of murine splenic activated T cells. (L and M) Mean fluorescence intensity (MFI) of Brilliant Ultraviolet 395-labelled PD-1 on murine splenic activated CD4 + (L) and CD8 + (M) T cells. (N and O) MFI of Alexa Fluor 700-labelled TNF-□ (N) and PerCP-Cy5.5-labelled IFN-γ (O) on murine splenic activated CD8+ T cells. (P and Q) MFI of APC-Cyanine 7-labelled PD-1 on human CD4 + (P) and CD8 + (Q) T cells activated by human T-activator Dynabeads™ for 2 days. (R and S) MFI of Brilliant Violet 650-labelled TNF-□ (R) and PE-labelled IFN-γ (S) expression on human CD8 + T cells activated by human T-activator Dynabeads™ for 2 days. (T) Representative photos of KPC cells stained with crystal violet after co-culturing with activated T cells for 48 hours. (U) Quantification of crystal violet-stained area from panel T using QuPath v.0.6.0. n=6 per group for panels A-F. n=5 and 4 per group for panels H-O and U, respectively. N=3 per group for panels P-S. Data are represented as mean ± SEM except for panels G and T. Unpaired two-tailed Student’s t-tests were used except for panels A, D, G, T, and U. Multiple unpaired t-test corrected by the Holm-Šidák method was used for panels A and D, while One-way ANOVA followed by Tukey’s post hoc tests for panel U. Statistical significance was defined as follows: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. See also Figures S3-S7 and Table S3-S4.

Article Snippet: Sections were then incubated with CD8 alpha (D4W2Z) Rabbit Monoclonal Antibody (Cell Signaling Technology, 98941) at a 1:400 dilution for 30 minutes at room temperature.

Techniques: Activation Assay, Fluorescence, Expressing, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Systemic Cysteine Elevation Sustains T-Cell Activation to Potentiate PD-1 Blockade

doi: 10.64898/2026.03.16.711688

Figure Lengend Snippet: (A) Schematic of KPC tumor-bearing mice. Created with BioRender.com. (B and C) Relative cysteine levels in serum (B) and KPC tumor tissue (C) of the KPC tumor-bearing mice receiving anti-PD-1 injections combined with PBS (n=5) or Cys (n=7) gavage for 3 or 4 weeks. (D) Relative tumor weight of the KPC tumor-bearing mice receiving anti-PD-1 injections combined with PBS (n=5) or Cys (n=7) gavage for 3 or 4 weeks. (E-J) Absolute number of tumor-infiltrated CD45 + (E), CD3 + (F), CD4 + (G), CD8 + (H), and activated CD4 + (I) and CD8 + (J) (define as CD25 or/and CD69 positive) T cells detected by flow cytometry from mice from panel A (n=4 per group). (K) Absolute number of tumor-infiltrated TNF-□ and IFN-γ double-positive CD8 + T cells analyzed by flow cytometry from mice from panel A (n=4 per group). (L and M) The proportions of activated CD4 + (L) and activated CD8 + (M) (defined as CD25 or/and CD69 positive) T cells in mesenteric lymph nodes detected by flow cytometry from mice from panel A (n=5 per group). (N) Anti-PD-1 responding ratio of KPC-tumor bearing mice under anti-PD-1 treatment with or without Cys gavage (each dot represents the responding ratio calculated from a single independent animal experiment). Data are represented as mean ± SEM, whereas *p<0.05 and **p<0.01 by unpaired two-tailed Student’s t-tests.

Article Snippet: Sections were then incubated with CD8 alpha (D4W2Z) Rabbit Monoclonal Antibody (Cell Signaling Technology, 98941) at a 1:400 dilution for 30 minutes at room temperature.

Techniques: Flow Cytometry, Two Tailed Test