PMC

a Diagram showing the experimental design and timeline of the depletion study. Abbreviations: Combo, combination strategy; Combo⁺, adapted combination strategy; i.c.m., intra-cisterna magna; i.p., intraperitoneal; sac, sacrifice. b Quantification by flow cytometry of the number of Ly6G^TdT+ neutrophils in the blood of PBS- and IL-1α-injected Ly6g^Cre-TdT::R26-TdT mice pretreated with either the Combo⁺ strategy, Combo strategy, isotype control antibodies or saline. Mice were killed at 24 h post-i.c.m. injection (n = 3 Saline + PBS, n = 5 Isotype control + PBS, n = 4 Combo + PBS, n = 3 Combo⁺ + PBS, n = 3 Saline + IL-1α, n = 7 Isotype control + IL-1α, n = 4 Combo + IL-1α, n = 3 Combo⁺ + IL-1α). c Quantification of Ly6G^TdT+ neutrophils that infiltrated the spinal cord at 24 h post-i.c.m. injection (n = 3 Saline + PBS, n = 6 Isotype control + PBS, n = 4 Combo + PBS, n = 3 Combo⁺ + PBS, n = 4 Saline + IL-1α, n = 7 Isotype control + IL-1α, n = 4 Combo + IL-1α, n = 3 Combo⁺ + IL-1α). d, e Confocal images showing Ly6G^TdT+ cells (red) and laminin (green) in the spinal cord of Ly6g^Cre-TdT::R26-TdT mice injected i.c.m. with rmIL-1α, and pretreated i.p. with either isotype control antibodies (d) or the Combo⁺ treatment (e). f, g Confocal immunofluorescence imaging of Olig2 and CC1 in the spinal cord white matter of Ly6g^Cre-TdT::R26-TdT mice injected i.c.m. with either IL-1α or PBS, and treated with either isotype control antibodies (f) or the Combo⁺ strategy (g). h Quantification of Olig2⁺ CC1⁺ mature OLs in the spinal cord white matter of mice (n = 3 Saline + PBS, n = 6 Isotype control + PBS, n = 4 Combo + PBS, n = 3 Combo⁺ + PBS, n = 4 Saline + IL-1α, n = 7 Isotype control + IL-1α, n = 4 Combo + IL-1α, n = 3 Combo⁺ + IL-1α). i Percentage and total number (in parentheses) of Ly6G^TdT+ neutrophils in the spinal cord gray matter (GM) versus white matter (WM). All data are mean values + /− SEM and statistical significance was determined by a two-way ANOVA followed by Bonferroni post-hoc test (b, c, h). Pairwise comparisons and p-values are indicated in graphs. Scale bars: (d, e, in e) 50 µm, (f, g, in g) 50 µm.

Ratiometric cellular uptake and release of dual drugs from Combo NP. Uptake of cisplatin and GMP in Combo NP, Sepa NP, and free drugs at 37 °C for 4 h in UMUC3 cells (A). Accumulative uptake of Combo NP loaded with cisplatin and GMP in UMUC3 Cells (B). In vitro release kinetics of cisplatin and GMP from Combo NP and single NP in PBS at 37 °C (C) and intracellular release of cisplatin and GMP from Combo NP (D). IC₅₀ of free GMP, cisplatin, and Combo free at molar ratio 5.3:1, as well as single drug NP and Combo NP at molar ratio 5.3:1 (E). X-axis indicated the total concentration of dual drugs or single drug formulations. The corresponding CI vs Fa plots of Combo NP and Combo free were shown (F). DL of cisplatin and GMP in Combo NP is 0.8 wt% and 4.6 wt% respectively, while DL of cisplatin and GMP in single NP is 4.4 wt% and 4.2 wt% respectively. n.s.: no significant difference; * P<0.05; ** P<0.01.

Effect of HBx on cell cycle progression. (A) Cell cycle distributions in HepG2 cells expressing WT and mutant HBx, with empty vector as a control. Upper panels: representative cell cycle plots. Lower panel: average percentage of cells in different phases of the cell cycle obtained from four analyses. Dot: S-phase in Combo mutant vs control Vector or TA, P<.01; asterisk: S-phase in Combo mutant vs WT HBx, P<.001. (B) Cotransfection of p21 abolished the S-phase stimulation by Combo mutant in Huh7 cells. Asterisk: p21 and Combo mutant cotransfection vs Combo mutant only, P<.01. (C) Dynamics of p21 expression and cell cycle progression in HBx transfected HepG2 cells. Upper figure: p21 expression at the indicated time points. Lower figure: cell cycle distributions at the same time points. (D) p21 silencing increased the proportion of S-phase in WT HBx expressing Huh7 cells. Asterisk: p21 siRNA vs nontargeting siRNA, P<.01. (E) Combo mutant accelerates S-phase progression. Upper figure: BrdU incorporation indices of synchronized HepG2 cells after serum release. Dot: Combo mutant vs TA or control Vector, P<.05; asterisk: Combo mutant vs WT, P<.01. Lower figure: BrdU incorporation indices of Combo mutant expressing Huh7 cells with and without p21 overexpression. Asterisk: p21 and Combo mutant cotransfection vs Combo mutant only, P<.05. Data represent means ± SD from quadruplicate. (F) Effect of WT and mutant HBx on cyclin E expression in PHH.

a Tumor growth of EMT6 tumor-bearing athymic nu/nu mice (PBS and ENT, n = 10; NHS-rmIL12, n = 8; combo, n = 9; P < 0.0001) treated with Entinostat (ENT) and/or NHS-rmIL12 as indicated in the schematic. b Tumor growth (P < 0.0001) and (c) survival (P < 0.0001) in syngeneic Balb/c mice after treatment with ENT+NHS-rmIL12 as in Fig.  with or without CD8 depletion, n = 9. Inset indicates median overall survival (mOS). For all tumor growth curves, gray shaded area indicates Entinostat treatment and dashed lines are NHS-rmIL12 doses. CD8 depletion period is indicated with orange shading. d Top 10 pathways downregulated in TAM gene clusters from CD8-depleted mice treated with combination therapy vs. undepleted. e Expression by scRNAseq of select M1 (Cd38, Nos2) and M2-associated (Mrc1, Arg1) genes in CD45⁺ cells isolated from CD8-depleted or undepleted EMT6 tumors. Each red dot represents one single cell. f Effect of CD8 depletion in the relative frequency of M1- and M2-like TAMs by scRNAseq. Mice from depletion study were analyzed 21 days after tumor implant and (g) CD8 tumor-infiltrating lymphocytes (TIL) (PBS, n = 6; combo, n = 4, combo/CD8dep, n = 5; P = 0.0028), (h) M1-like CD38⁺ CD206^- TAMs (PBS and combo/CD8dep^, n = 6^; combo, n = 5; P < 0.0001), (i) M2-like CD38^-CD206⁺ TAMs (PBS and combo/CD8dep, n = 6; combo, n = 5; P < 0.0001), and (j) the ratio of M1/M2 TAMs using (i) CD38/CD206 (PBS and combo/CD8dep, n = 6; combo, n = 5; P = 0.0030) or (ii) NOS2/Arg1 (PBS and combo/CD8dep, n = 6; combo, n = 4; P < 0.0001) was quantified by flow cytometry. k–l Graphs show quantification of (k) IL12p70 (PBS and combo, n = 10; ENT and NHS-rmIL12, n = 6; combo/CD8dep, n = 12; P < 0.0001), TNFα (PBS, n = 11; ENT and NHS-rmIL12, n = 6; combo, n = 8; combo/CD8dep, n = 12; P = 0.0011), IFNγ (PBS, n = 10; ENT, n = 6; NHS-rmIL12, n = 5; combo, n = 11; combo/CD8dep, n = 12; P = 0.0002), and (l) IFNβ (PBS, n = 11; ENT and NHS-rmIL12, n = 6; combo, n = 10; combo/CD8dep, n = 12; P = 0.0009), GM-CSF (PBS, n = 11; ENT and NHS-rmIL12, n = 6; combo, n = 11; combo/CD8dep, n = 12; P = 0.0005), IL1α (PBS, n = 12; ENT and NHS-rmIL12, n = 6; combo, n = 10; combo/CD8dep, n = 12; P = 0.0009), and IL1β (PBS, n = 12; ENT and NHS-rmIL12, n = 6; combo, n = 11; combo/CD8dep, n = 12; P = 0.0004) in tumor microenvironment (TME) supernatant on day 21 post-tumor implant in EMT6 tumor-bearing Balb/c mice. m, n Effect of EMT6 tumor CD8 depletion on designated chemokines’ (m) gene expression by RNAseq and (n) CCL3 (P = 0.0021), CCL4 (P = 0.0146), CCL5 (P = 0.0007), CXCL9 (P = 0.0014), and CXCL10 (P = 0.0002) protein levels in TME supernatant (PBS, n = 11; ENT, n = 6; NHS-rmIL12, n = 5; combo and combo/CD8dep, n = 10). Each dot represents one single cell (e). Correlation plots show values from individual mice. Truncated violin plots show values from individual mice with contours denoting kernel density distributions; dashed line, median; and dotted line, interquartile range. For survival, log-rank (Mantel–Cox) was used for comparisons. One-way ANOVA with Tukey’s multiple comparisons test was used for all comparisons except two-way ANOVA was used for average tumor growth comparison. Statistics are all two-sided. Grey = p < 0.05, red = p < 0.01, blue = p < 0.001, black = p < 0.0001. Data from single independent experiments (b–f, m) or pooled from two experiments conducted independently with similar results (a, g–l, n). Combo, ENT+NHS-rmIL12. Source data are provided as a Source Data file.