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flow cytometry pe anti mouse ebi3  (R&D Systems)


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    R&D Systems flow cytometry pe anti mouse ebi3
    ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + <t>EBI3</t> + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
    Flow Cytometry Pe Anti Mouse Ebi3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+ebi3/bio_rxiv__64898__2026__02__23__707416-190-6-17?v=R%26D+Systems
    Average 93 stars, based on 2 article reviews
    flow cytometry pe anti mouse ebi3 - by Bioz Stars, 2026-08
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    Images

    1) Product Images from "IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis"

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    Journal: bioRxiv

    doi: 10.64898/2026.02.23.707416

    ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
    Figure Legend Snippet: ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Techniques Used: Expressing, Infection, Flow Cytometry, Quantitative RT-PCR, Staining, Immunoprecipitation, Western Blot, Control, Enzyme-linked Immunosorbent Assay

    ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.
    Figure Legend Snippet: ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

    Techniques Used: ChIP-qPCR, Infection, Pull Down Assay, Western Blot, Binding Assay, Labeling, Control, Expressing, Transfection, Flow Cytometry

    ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
    Figure Legend Snippet: ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Techniques Used: Adoptive Transfer Assay, Transfection, Control, Infection, Expressing, Flow Cytometry

    LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .
    Figure Legend Snippet: LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .

    Techniques Used: Binding Assay, Derivative Assay, Activation Assay, Expressing, In Vivo



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    Rockland Immunochemicals rat anti mouse ebi3
    ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + <t>EBI3</t> + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.
    Rat Anti Mouse Ebi3, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rat anti-mouse ebi3  (Rockland Immunochemicals)
    90
    Rockland Immunochemicals rat anti-mouse ebi3
    M2 macrophages promoted the expression of IL-35 and TGF-β1 in B cells. B cells were co-cultured with macrophages for 48 h, followed by flow cytometry and immunofluorescence staining detection. ( A ) A schematic of B cells co-cultured with macrophages. ( B ) IL-35 <t>(Ebi3</t> + IL-12a + ) expression in B cells detected by flow cytometry. ( C ) Statistical data of IL-35 expression in B cells ( n = 12). ( D ) IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( E ) Statistical data of IL-35 expression in B cells ( n = 7). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 12). ( H ) IL-10 expression in B cells detected by flow cytometry. ( I ) Statistical data of IL-10 expression in B cells. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.
    Rat Anti Mouse Ebi3, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse il  (R&D Systems)
    93
    R&D Systems mouse il
    M2 macrophages promoted the expression of IL-35 and TGF-β1 in B cells. B cells were co-cultured with macrophages for 48 h, followed by flow cytometry and immunofluorescence staining detection. ( A ) A schematic of B cells co-cultured with macrophages. ( B ) IL-35 <t>(Ebi3</t> + IL-12a + ) expression in B cells detected by flow cytometry. ( C ) Statistical data of IL-35 expression in B cells ( n = 12). ( D ) IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( E ) Statistical data of IL-35 expression in B cells ( n = 7). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 12). ( H ) IL-10 expression in B cells detected by flow cytometry. ( I ) Statistical data of IL-10 expression in B cells. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.
    Mouse Il, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ebi3  (Proteintech)
    93
    Proteintech ebi3
    The expression of IL35 is increased in human HBV-related HCC tissues. (A , B) The expression of <t>EBI3</t> and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by IHC staining, and the bar chart represents the ratio of staining intensity between cancerous tissues and adjacent tissues. (C , D) The mRNA expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by RT-qPCR, and the bar charts represent their expression differences between cancerous tissues and adjacent tissues. (E) The protein expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by Western blot, and the bar charts represent their expression differences between cancerous tissues and adjacent tissues.The results are expressed as the mean ± SD of three experiments, * P < 0.05 compared with the normal group.
    Ebi3, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

    Techniques: Expressing, Infection, Flow Cytometry, Quantitative RT-PCR, Staining, Immunoprecipitation, Western Blot, Control, Enzyme-linked Immunosorbent Assay

    ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

    Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

    Techniques: ChIP-qPCR, Infection, Pull Down Assay, Western Blot, Binding Assay, Labeling, Control, Expressing, Transfection, Flow Cytometry

    ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

    Techniques: Adoptive Transfer Assay, Transfection, Control, Infection, Expressing, Flow Cytometry

    LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .

    Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

    Techniques: Binding Assay, Derivative Assay, Activation Assay, Expressing, In Vivo

    ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Article Snippet: The following antibodies were used for confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

    Techniques: Expressing, Infection, Flow Cytometry, Quantitative RT-PCR, Staining, Immunoprecipitation, Western Blot, Control, Enzyme-linked Immunosorbent Assay

    ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

    Article Snippet: The following antibodies were used for confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

    Techniques: ChIP-qPCR, Infection, Pull Down Assay, Western Blot, Binding Assay, Labeling, Control, Expressing, Transfection, Flow Cytometry

    ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

    Article Snippet: The following antibodies were used for confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

    Techniques: Adoptive Transfer Assay, Transfection, Control, Infection, Expressing, Flow Cytometry

    LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .

    Journal: bioRxiv

    Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

    doi: 10.64898/2026.02.23.707416

    Figure Lengend Snippet: LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .

    Article Snippet: The following antibodies were used for confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

    Techniques: Binding Assay, Derivative Assay, Activation Assay, Expressing, In Vivo

    M2 macrophages promoted the expression of IL-35 and TGF-β1 in B cells. B cells were co-cultured with macrophages for 48 h, followed by flow cytometry and immunofluorescence staining detection. ( A ) A schematic of B cells co-cultured with macrophages. ( B ) IL-35 (Ebi3 + IL-12a + ) expression in B cells detected by flow cytometry. ( C ) Statistical data of IL-35 expression in B cells ( n = 12). ( D ) IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( E ) Statistical data of IL-35 expression in B cells ( n = 7). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 12). ( H ) IL-10 expression in B cells detected by flow cytometry. ( I ) Statistical data of IL-10 expression in B cells. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.

    Journal: International Journal of Molecular Sciences

    Article Title: Pro-Resolving Macrophage-Induced IL-35 + but Not TGF-β1 + Regulatory B Cell Activation Requires the PD-L1/PD-1 Pathway

    doi: 10.3390/ijms26115332

    Figure Lengend Snippet: M2 macrophages promoted the expression of IL-35 and TGF-β1 in B cells. B cells were co-cultured with macrophages for 48 h, followed by flow cytometry and immunofluorescence staining detection. ( A ) A schematic of B cells co-cultured with macrophages. ( B ) IL-35 (Ebi3 + IL-12a + ) expression in B cells detected by flow cytometry. ( C ) Statistical data of IL-35 expression in B cells ( n = 12). ( D ) IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( E ) Statistical data of IL-35 expression in B cells ( n = 7). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 12). ( H ) IL-10 expression in B cells detected by flow cytometry. ( I ) Statistical data of IL-10 expression in B cells. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.

    Article Snippet: Cells were stained with rat anti-mouse EBi3 (1:150, cat: 210-501-B66, Rockland, ThermoFisher Scientific, Waltham, MA, USA) and rabbit anti-mouse IL-12a (1:100, cat: BS-0767R, Bioss, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4 °C, and the Alexa flour 488-labeled goat anti-rabbit IgG (1:500) and Alexa flour 594-labeled goat anti-rat IgG (1:500) secondary antibodies were used to recognize the primary antibody.

    Techniques: Expressing, Cell Culture, Flow Cytometry, Immunofluorescence, Staining

    M2 macrophage-induced IL-35 and TGF-β1 expression in B cells requires direct cell–cell contact. B cells were co-cultured with M2 macrophages with or without a trans-well insert, followed by flow cytometry and immunofluorescence staining. ( A ) A schematic of B cells co-cultured with macrophages with a trans-well insert. ( B ) A typical picture of IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( C ) Statistical data of IL-35 expression in B cells detected by immunofluorescence staining ( n = 7). ( D ) IL-35 (Ebi3 + IL-12a + ) expression in B cells detected by flow cytometry. ( E ) Statistical data of IL-35 expression in B cells detected by flow cytometry ( n = 6). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 6). Statistical significance was analyzed using a one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.

    Journal: International Journal of Molecular Sciences

    Article Title: Pro-Resolving Macrophage-Induced IL-35 + but Not TGF-β1 + Regulatory B Cell Activation Requires the PD-L1/PD-1 Pathway

    doi: 10.3390/ijms26115332

    Figure Lengend Snippet: M2 macrophage-induced IL-35 and TGF-β1 expression in B cells requires direct cell–cell contact. B cells were co-cultured with M2 macrophages with or without a trans-well insert, followed by flow cytometry and immunofluorescence staining. ( A ) A schematic of B cells co-cultured with macrophages with a trans-well insert. ( B ) A typical picture of IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( C ) Statistical data of IL-35 expression in B cells detected by immunofluorescence staining ( n = 7). ( D ) IL-35 (Ebi3 + IL-12a + ) expression in B cells detected by flow cytometry. ( E ) Statistical data of IL-35 expression in B cells detected by flow cytometry ( n = 6). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 6). Statistical significance was analyzed using a one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.

    Article Snippet: Cells were stained with rat anti-mouse EBi3 (1:150, cat: 210-501-B66, Rockland, ThermoFisher Scientific, Waltham, MA, USA) and rabbit anti-mouse IL-12a (1:100, cat: BS-0767R, Bioss, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4 °C, and the Alexa flour 488-labeled goat anti-rabbit IgG (1:500) and Alexa flour 594-labeled goat anti-rat IgG (1:500) secondary antibodies were used to recognize the primary antibody.

    Techniques: Expressing, Cell Culture, Flow Cytometry, Immunofluorescence, Staining

    The expression of IL35 is increased in human HBV-related HCC tissues. (A , B) The expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by IHC staining, and the bar chart represents the ratio of staining intensity between cancerous tissues and adjacent tissues. (C , D) The mRNA expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by RT-qPCR, and the bar charts represent their expression differences between cancerous tissues and adjacent tissues. (E) The protein expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by Western blot, and the bar charts represent their expression differences between cancerous tissues and adjacent tissues.The results are expressed as the mean ± SD of three experiments, * P < 0.05 compared with the normal group.

    Journal: Scientific Reports

    Article Title: IL35 modulates HBV-related HCC progression via IL6-STAT3 signaling

    doi: 10.1038/s41598-025-89015-y

    Figure Lengend Snippet: The expression of IL35 is increased in human HBV-related HCC tissues. (A , B) The expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by IHC staining, and the bar chart represents the ratio of staining intensity between cancerous tissues and adjacent tissues. (C , D) The mRNA expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by RT-qPCR, and the bar charts represent their expression differences between cancerous tissues and adjacent tissues. (E) The protein expression of EBI3 and p35 in human HBV-related HCC tissues and adjacent peritumoral tissues was detected by Western blot, and the bar charts represent their expression differences between cancerous tissues and adjacent tissues.The results are expressed as the mean ± SD of three experiments, * P < 0.05 compared with the normal group.

    Article Snippet: Subsequently, the PVDF membranes were blocked with 5% BSA and incubated with the following primary antibodies overnight at 4 °C: EBI3 (Protein-tech, Cat#12371-1-AP), p35 (Abcam, Cat#ab131039), Bcl2 (Wanleibio, Cat#WL01556), Bax (Wanleibio, Cat#WL01637), IL6 (Wanleibio, Cat#WL02841), STAT3 (Santa Cruz, Cat#sc-8019), pSTAT3 (Santa Cruz, Cat#sc-8059), Histone-H3(Proteintect, Cat# 17168-1-AP),β-Actin (Bioworld, Cat#BS6007M).

    Techniques: Expressing, Immunohistochemistry, Staining, Quantitative RT-PCR, Western Blot

    The expression of IL35 is increased in the HBV-induced hepatoma cell model. (A , B) The mRNA and protein expression of EBI3 and p35 in HepG2(as control) and HepG2.2.15 cells was detected by RT-qPCR and Western blot respectively, and the bar charts represent their expression differences between two cells. (C , D) The mRNA and protein expression of EBI3 and p35 in HBV-induced HepG2 cells (pBLUE as control) were detected by RT-qPCR and Western blot respectively, and the bar charts represent their expression differences between two cells. (E , F) The mRNA expression of EBI3 and p35 in HBV-induced HepG2/Huh7 cells (pEGFP-N1 as control) were detected by RT-qPCR, and the bar charts represent their expression differences among the six groups. (G , H) The secretion of IL35 and IL6 in HBx-induced HepG2/Huh7 cells (pEGFP-N1 as control) were detected by ELISA, and the bar charts represent their expression differences among the three groups.The results are expressed as the mean ± SD of three experiments, * P < 0.05 compared with the control group.

    Journal: Scientific Reports

    Article Title: IL35 modulates HBV-related HCC progression via IL6-STAT3 signaling

    doi: 10.1038/s41598-025-89015-y

    Figure Lengend Snippet: The expression of IL35 is increased in the HBV-induced hepatoma cell model. (A , B) The mRNA and protein expression of EBI3 and p35 in HepG2(as control) and HepG2.2.15 cells was detected by RT-qPCR and Western blot respectively, and the bar charts represent their expression differences between two cells. (C , D) The mRNA and protein expression of EBI3 and p35 in HBV-induced HepG2 cells (pBLUE as control) were detected by RT-qPCR and Western blot respectively, and the bar charts represent their expression differences between two cells. (E , F) The mRNA expression of EBI3 and p35 in HBV-induced HepG2/Huh7 cells (pEGFP-N1 as control) were detected by RT-qPCR, and the bar charts represent their expression differences among the six groups. (G , H) The secretion of IL35 and IL6 in HBx-induced HepG2/Huh7 cells (pEGFP-N1 as control) were detected by ELISA, and the bar charts represent their expression differences among the three groups.The results are expressed as the mean ± SD of three experiments, * P < 0.05 compared with the control group.

    Article Snippet: Subsequently, the PVDF membranes were blocked with 5% BSA and incubated with the following primary antibodies overnight at 4 °C: EBI3 (Protein-tech, Cat#12371-1-AP), p35 (Abcam, Cat#ab131039), Bcl2 (Wanleibio, Cat#WL01556), Bax (Wanleibio, Cat#WL01637), IL6 (Wanleibio, Cat#WL02841), STAT3 (Santa Cruz, Cat#sc-8019), pSTAT3 (Santa Cruz, Cat#sc-8059), Histone-H3(Proteintect, Cat# 17168-1-AP),β-Actin (Bioworld, Cat#BS6007M).

    Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

    IL35 silencing promotes apoptosis of HBx-induced hepatoma cell lines. (A , B) The mRNA expression of EBI3 and p35 in HBx-induced HepG2/Huh7 cells transfected with siIL35 and siNC were detected by RT-qPCR, and the bar charts represent their expression differences between the two groups. (C , D) The apoptosis rates of HBx-induced HepG2/Huh7 cells transfected with siIL35 and siNC were detected by FCM with Annexin V-FITC/PI double staining, and Q1-LR and Q1-UR represent the proportion of early and late apoptotic cells respectively, and the bar charts represent the differences in apoptosis rate among the three groups. (E , F) The protein expression of apoptosis-related proteins in HBx-induced HepG2/Huh7 cells transfected with siIL35 and siNC was detected by Western blot, and the bar charts represent the differences in Bax/Bcl2 ratio among the three groups. The results are expressed as the mean ± SD of three experiments, * P < 0.05 compared with the siNC group.

    Journal: Scientific Reports

    Article Title: IL35 modulates HBV-related HCC progression via IL6-STAT3 signaling

    doi: 10.1038/s41598-025-89015-y

    Figure Lengend Snippet: IL35 silencing promotes apoptosis of HBx-induced hepatoma cell lines. (A , B) The mRNA expression of EBI3 and p35 in HBx-induced HepG2/Huh7 cells transfected with siIL35 and siNC were detected by RT-qPCR, and the bar charts represent their expression differences between the two groups. (C , D) The apoptosis rates of HBx-induced HepG2/Huh7 cells transfected with siIL35 and siNC were detected by FCM with Annexin V-FITC/PI double staining, and Q1-LR and Q1-UR represent the proportion of early and late apoptotic cells respectively, and the bar charts represent the differences in apoptosis rate among the three groups. (E , F) The protein expression of apoptosis-related proteins in HBx-induced HepG2/Huh7 cells transfected with siIL35 and siNC was detected by Western blot, and the bar charts represent the differences in Bax/Bcl2 ratio among the three groups. The results are expressed as the mean ± SD of three experiments, * P < 0.05 compared with the siNC group.

    Article Snippet: Subsequently, the PVDF membranes were blocked with 5% BSA and incubated with the following primary antibodies overnight at 4 °C: EBI3 (Protein-tech, Cat#12371-1-AP), p35 (Abcam, Cat#ab131039), Bcl2 (Wanleibio, Cat#WL01556), Bax (Wanleibio, Cat#WL01637), IL6 (Wanleibio, Cat#WL02841), STAT3 (Santa Cruz, Cat#sc-8019), pSTAT3 (Santa Cruz, Cat#sc-8059), Histone-H3(Proteintect, Cat# 17168-1-AP),β-Actin (Bioworld, Cat#BS6007M).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Double Staining, Western Blot