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Revvity pstat1 tyr701

T reg cells in VV-resistant tumors have elevated TGFβ and a repressed response to IFNγ. (A) Luminex cytokine analysis of TIF harvested from untreated MEER vvR or MEER vvS implanted in C57BL/6 mice. Three mice per group, three technical repeats per mouse. (B) Active TGFβ 1–3 concentration in the TIF of untreated MEER vvS or MEER vvR tumors as determined by TGFβ reporter assay. (C) TGFβR2 expression on CD8 + , Foxp3− T conv , or Foxp3+ T reg cells in untreated MEER vvR or MEER vvS implanted in Foxp3-reporter mice. Representative histograms from a MEER vvR tumor. 4 repeats, 10 M vvS , 9 M vvR mice. (D) Experimental schema of E–L and Q. Repeated three times. (E–L and Q) Percentage of (E) LAP-TGFβ1+, (F) GARP+, (G) CD103+, (I) TCF1+, (J) CD25 + , (K) CD122+, (L) PD-1+ Tim3+, and percentage and MFI of (H) Nrp1+ and (Q) <t>pSTAT1+</t> T reg cells by flow cytometry as in D. (M) IFNγ concentration in TIF of MEER vvR and MEER vvS at 7 d after treatment with PBS or VV as in . (N–P) Percentage of pSTAT1 S727 + (N) CD8 + cells, (O) T conv cells, and (P) T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . (R) Representative flow plots and percentage of IFNγ+ T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . Cells were restimulated with PMA/ionomycin direct ex vivo from tumors. M vvS = MEER vvS , M vvR = MEER vvR . Data represent two (A, B, and I–M), three (C, E–H, and Q), or four (N–P and R) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test (A), unpaired T test (B), one-way ANOVA with Sidak’s multiple comparison test paired T test (C, M–P, and R), or paired t test (E–L and Q). ns, non-significant. Error bars indicate SEMs.

Pstat1 Tyr701, supplied by Revvity, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pstat1 tyr701 - by Bioz Stars, 2026-06

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1) Product Images from "An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment"

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20230053

Figure Legend Snippet: T reg cells in VV-resistant tumors have elevated TGFβ and a repressed response to IFNγ. (A) Luminex cytokine analysis of TIF harvested from untreated MEER vvR or MEER vvS implanted in C57BL/6 mice. Three mice per group, three technical repeats per mouse. (B) Active TGFβ 1–3 concentration in the TIF of untreated MEER vvS or MEER vvR tumors as determined by TGFβ reporter assay. (C) TGFβR2 expression on CD8 + , Foxp3− T conv , or Foxp3+ T reg cells in untreated MEER vvR or MEER vvS implanted in Foxp3-reporter mice. Representative histograms from a MEER vvR tumor. 4 repeats, 10 M vvS , 9 M vvR mice. (D) Experimental schema of E–L and Q. Repeated three times. (E–L and Q) Percentage of (E) LAP-TGFβ1+, (F) GARP+, (G) CD103+, (I) TCF1+, (J) CD25 + , (K) CD122+, (L) PD-1+ Tim3+, and percentage and MFI of (H) Nrp1+ and (Q) pSTAT1+ T reg cells by flow cytometry as in D. (M) IFNγ concentration in TIF of MEER vvR and MEER vvS at 7 d after treatment with PBS or VV as in . (N–P) Percentage of pSTAT1 S727 + (N) CD8 + cells, (O) T conv cells, and (P) T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . (R) Representative flow plots and percentage of IFNγ+ T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . Cells were restimulated with PMA/ionomycin direct ex vivo from tumors. M vvS = MEER vvS , M vvR = MEER vvR . Data represent two (A, B, and I–M), three (C, E–H, and Q), or four (N–P and R) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test (A), unpaired T test (B), one-way ANOVA with Sidak’s multiple comparison test paired T test (C, M–P, and R), or paired t test (E–L and Q). ns, non-significant. Error bars indicate SEMs.

Techniques Used: Luminex, Concentration Assay, Reporter Assay, Expressing, Flow Cytometry, Ex Vivo, Comparison

Phenotyping observed in TIL T reg cells is found in dLN and TIL T conv to a lesser extent. (A–I) Quantification of the percentages of dLN T reg cells from paired tumors in Foxp3-Ametrine or Foxp3-RFP mice as in . (A) LAP-TGFβ1+, (B) GARP+, (C) CD103+, (D) Nrp1+, (E) TCF1+, (F) CD25 + , (G) CD122+, (H) PD-1+ Tim3+, and (I) pSTAT1+. (J–Q) Representative flow plots and quantification of T reg phenotyping markers on tumor-infiltrating T conv cells as in . Representative histograms of tumor-infiltrating T conv (Foxp3−) and T reg (Foxp3+) cells are shown for comparison, and quantification axes are scaled for T reg expression. Quantification and flow plots of (J) LAP-TGFβ1+, (K) GARP+, (L) CD103+, (M) Nrp1+, (N) CD25 + , (O) CD122+, (P) Tim3, and PD-1 (Q) pSTAT1+ Tconv cells. (R–U) Representative flow plots and quantification in paired dLN and tumors as in of (R) CD62L and CD44 on T conv cells, (S) CD62L and CD44 on CD8 + cells, (T) TCF1 on T conv cells, and (U) TCF1 on CD8 + cells. CM, central memory CD62L+ CD44 + ; Eff, effector CD62L− CD44 + ; Naïve, CD62L+ CD44 − . Data represent two (R–U) or three (A–Q) independent experiments. Each point represents an individual mouse *P < 0.05, **P < 0.01, ***P < 0.001 by paired T test (A–Q, T, and U) or one way ANOVA with Sidaks multiple comparisons test (R and S). ns, non-significant. Error bars indicate SEMs.

Figure Legend Snippet: Phenotyping observed in TIL T reg cells is found in dLN and TIL T conv to a lesser extent. (A–I) Quantification of the percentages of dLN T reg cells from paired tumors in Foxp3-Ametrine or Foxp3-RFP mice as in . (A) LAP-TGFβ1+, (B) GARP+, (C) CD103+, (D) Nrp1+, (E) TCF1+, (F) CD25 + , (G) CD122+, (H) PD-1+ Tim3+, and (I) pSTAT1+. (J–Q) Representative flow plots and quantification of T reg phenotyping markers on tumor-infiltrating T conv cells as in . Representative histograms of tumor-infiltrating T conv (Foxp3−) and T reg (Foxp3+) cells are shown for comparison, and quantification axes are scaled for T reg expression. Quantification and flow plots of (J) LAP-TGFβ1+, (K) GARP+, (L) CD103+, (M) Nrp1+, (N) CD25 + , (O) CD122+, (P) Tim3, and PD-1 (Q) pSTAT1+ Tconv cells. (R–U) Representative flow plots and quantification in paired dLN and tumors as in of (R) CD62L and CD44 on T conv cells, (S) CD62L and CD44 on CD8 + cells, (T) TCF1 on T conv cells, and (U) TCF1 on CD8 + cells. CM, central memory CD62L+ CD44 + ; Eff, effector CD62L− CD44 + ; Naïve, CD62L+ CD44 − . Data represent two (R–U) or three (A–Q) independent experiments. Each point represents an individual mouse *P < 0.05, **P < 0.01, ***P < 0.001 by paired T test (A–Q, T, and U) or one way ANOVA with Sidaks multiple comparisons test (R and S). ns, non-significant. Error bars indicate SEMs.

Techniques Used: Comparison, Expressing

TGFβ limits IFNγ signaling and increases T reg cell stability. (A) Immunoblot and densitometry of pSTAT1 Y701 , STAT1, and β-actin in T reg cells sorted from spleen and lymph node of a Foxp3 reporter mouse and treated for 30 min with IFNγ, TGFβ1, or both. (B) Quantification and Cell Trace Violet (CTV) plots of the proliferation of stimulated Thy1.1+ CD4 responder cells in the presence of suppressing T reg cells at the 1:8 T reg cell:responder ratio in an in vitro suppression assay. Percent suppression is normalized to the proliferation index of stimulated CD4 + responder control without T reg cells. T reg cells were sorted from spleen and lymph node of a Foxp3-reporter mouse and then cultured for 3 d in IFNγ, TGFβ, or both. Cells were then sorted again to purify Foxp3+ T reg cells and then co-cultured in the suppression assay with CTV-labeled responder CD4 + cells. (C) Surface Nrp1 expression on sorted T reg cells from spleen and lymph node of a Foxp3-reporter mouse cultured in vitro in varying TGFβ concentrations for 48 h (D–F) An EV control and TGFβ 1 overexpressing (TGFβ OE) line were generated from the MEER vvS line. (D) Tumor growth of C57BL/6 mice implanted intradermally with MEER vvS-EV or MEER vvS-TGFβ OE and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (E) Survival of D. (F) Average tumor growth of MEER vvS-EV and MEER vvS-TGFβ OE as in D. Data represent two (C), four (A and D–F), or five (B) independent experiments; each point or line represents an individual mouse (A–D). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Sidak’s multiple comparison test (A and C), paired T test (B), Mantel-Cox test (E), or mixed effects analysis (F). ns, non-significant. Error bars indicate SEMs. Source data are available for this figure: .

Figure Legend Snippet: TGFβ limits IFNγ signaling and increases T reg cell stability. (A) Immunoblot and densitometry of pSTAT1 Y701 , STAT1, and β-actin in T reg cells sorted from spleen and lymph node of a Foxp3 reporter mouse and treated for 30 min with IFNγ, TGFβ1, or both. (B) Quantification and Cell Trace Violet (CTV) plots of the proliferation of stimulated Thy1.1+ CD4 responder cells in the presence of suppressing T reg cells at the 1:8 T reg cell:responder ratio in an in vitro suppression assay. Percent suppression is normalized to the proliferation index of stimulated CD4 + responder control without T reg cells. T reg cells were sorted from spleen and lymph node of a Foxp3-reporter mouse and then cultured for 3 d in IFNγ, TGFβ, or both. Cells were then sorted again to purify Foxp3+ T reg cells and then co-cultured in the suppression assay with CTV-labeled responder CD4 + cells. (C) Surface Nrp1 expression on sorted T reg cells from spleen and lymph node of a Foxp3-reporter mouse cultured in vitro in varying TGFβ concentrations for 48 h (D–F) An EV control and TGFβ 1 overexpressing (TGFβ OE) line were generated from the MEER vvS line. (D) Tumor growth of C57BL/6 mice implanted intradermally with MEER vvS-EV or MEER vvS-TGFβ OE and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (E) Survival of D. (F) Average tumor growth of MEER vvS-EV and MEER vvS-TGFβ OE as in D. Data represent two (C), four (A and D–F), or five (B) independent experiments; each point or line represents an individual mouse (A–D). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Sidak’s multiple comparison test (A and C), paired T test (B), Mantel-Cox test (E), or mixed effects analysis (F). ns, non-significant. Error bars indicate SEMs. Source data are available for this figure: .

Techniques Used: Western Blot, In Vitro, Suppression Assay, Control, Cell Culture, Labeling, Expressing, Generated, Injection, Comparison

VVdnTGFbmm only affects T reg cell phenotype in the tumor. (A) Western blot for TGFβ in EV control and TGFβ 1 overexpressing (TGFβ OE) MEER vvS lines and MEER vvR and MEER vvS as in . (B) Active TGFβ 1–3 levels measured in TIF of LLC, MC38 (colon adenocarcinoma), B16-F10 (melanoma), and C24 ( Pten –/– Braf V600E melanoma) tumors in C57Bl/6 mice as in . The average TGFβ concentration of MEER vvS (light blue) and MEER vvR (gray) from are overlaid as dotted lines. (C) Growth curve of LLC tumors treated with PBS or VV (black arrowhead) as in . (D–N) Representative flow plots and quantification of dLN (D–K) and T reg phenotyping markers on tumor infiltrating T conv cells (L–N) in Foxp3-Ametrine or Foxp3-RFP mice as in . Quantification of dLN (D) percent Foxp3+ of CD4 + and (E) MFI of Foxp3. Quantification and representative flow plots of (F) Nrp1+, (G) pSTAT1+, and (H) LAP-TGFβ1 on dLN T reg cells. Quantification of dLN (I) percent Foxp3− of CD4 + and CD8 + . Quantification and representative flow plots of (J) TNFα and IFNγ in T conv cells with direct ex vivo PMA/ionomycin stimulation and (K) PD-1 and Tim3 on CD8 + cells in dLN. Quantification and representative flow plots of (L) Nrp1+, (M) pSTAT1+, and (N) LAP-TGFβ1 on TIL T conv cells. Data represent two (A–C) or four (D–N) independent experiments. Each dot or line represents a technical repeat (A) or mouse (B–N). *P < 0.05 by one-way ANOVA with Sidak’s multiple comparisons test. ns, non-significant. Source data are available for this figure: .

Figure Legend Snippet: VVdnTGFbmm only affects T reg cell phenotype in the tumor. (A) Western blot for TGFβ in EV control and TGFβ 1 overexpressing (TGFβ OE) MEER vvS lines and MEER vvR and MEER vvS as in . (B) Active TGFβ 1–3 levels measured in TIF of LLC, MC38 (colon adenocarcinoma), B16-F10 (melanoma), and C24 ( Pten –/– Braf V600E melanoma) tumors in C57Bl/6 mice as in . The average TGFβ concentration of MEER vvS (light blue) and MEER vvR (gray) from are overlaid as dotted lines. (C) Growth curve of LLC tumors treated with PBS or VV (black arrowhead) as in . (D–N) Representative flow plots and quantification of dLN (D–K) and T reg phenotyping markers on tumor infiltrating T conv cells (L–N) in Foxp3-Ametrine or Foxp3-RFP mice as in . Quantification of dLN (D) percent Foxp3+ of CD4 + and (E) MFI of Foxp3. Quantification and representative flow plots of (F) Nrp1+, (G) pSTAT1+, and (H) LAP-TGFβ1 on dLN T reg cells. Quantification of dLN (I) percent Foxp3− of CD4 + and CD8 + . Quantification and representative flow plots of (J) TNFα and IFNγ in T conv cells with direct ex vivo PMA/ionomycin stimulation and (K) PD-1 and Tim3 on CD8 + cells in dLN. Quantification and representative flow plots of (L) Nrp1+, (M) pSTAT1+, and (N) LAP-TGFβ1 on TIL T conv cells. Data represent two (A–C) or four (D–N) independent experiments. Each dot or line represents a technical repeat (A) or mouse (B–N). *P < 0.05 by one-way ANOVA with Sidak’s multiple comparisons test. ns, non-significant. Source data are available for this figure: .

Techniques Used: Western Blot, Control, Concentration Assay, Ex Vivo

C24 melanoma model has a similar phenotype to MEER vvR after VV treatment. (A) Tumor growth (left, middle) and survival (right) of C57BL/6 mice implanted intradermally with C24 and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (B) Foxp3-Ametrine or Fpxp3-RFP reporter mice implanted intradermally with C24 were as in A. Tumors and lymph nodes were harvested 7 d after treatment for phenotypic analysis. Percentage and total counts of CD8 + T cells and the ratio of T conv cells to T reg cells in treated tumors. (C) Percentage of Foxp3+ CD4 + T conv cells. (D) PD-1 and Tim3 expression on CD8 + cells. (E and F) (E) Production of granzyme B in CD8 + T cells and (F) MFI of pSTAT1 on T conv , CD8 + , and T reg cells as in B. (G) Production of IFNγ in T reg cells as in B after restimulation with PMA and ionomycin as in B. (H) IFNγ measured by ELISA from the TIL of CL24 untreated and 7 d after VV treatment as in B. Mice were implanted with C24 and treated with VV dnTGFβmm and αPD-1 as in . At day 8 after VV treatment, after three doses of αPD-1 were received, tumors were harvested for TIL analysis. (I–N) Representative flow plots and quantifications of (I) IFNγ+ T reg cells, (J) IFNγ + T conv cells, (K) IFNγ + CD8 + cells, (L) Tim3+ PD-1+ T reg cells, (M) TCF1+ T reg cells, and (N) TCF1+ CD8 + cells. Cytokine analysis was performed direct ex vivo with PMA and ionomycin restimulation. (O) Schematic for P and Q. (P) Tumor growth and survival (bottom) of C57BL/6 mice implanted intradermally with bilateral C24 and, when tumors were ∼20 mm 2 , one was treated with a single IT injection of VV dnTGFbmm (injected) at 2.5 × 10 6 PFU/mouse (black arrowhead). Mice were sacrificed when either tumor reached 15 mm in any direction. Starting at 4 d after VV or PBS treatment, mice were given anti-PD-1 or isotype control IP three times a week. (Q) Average growth of H. Data represent two independent experiments. Each point or line represents an individual mouse (A–P). *P < 0.05, **P < 0.01, ****P < 0.0001 by Welch’s T test (B–H), Mantel-Cox test (A and P), unpaired t Test (I–N), or mixed-effects analysis (Q). ns, non-significant. Error bars indicate SEMs.

Figure Legend Snippet: C24 melanoma model has a similar phenotype to MEER vvR after VV treatment. (A) Tumor growth (left, middle) and survival (right) of C57BL/6 mice implanted intradermally with C24 and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (B) Foxp3-Ametrine or Fpxp3-RFP reporter mice implanted intradermally with C24 were as in A. Tumors and lymph nodes were harvested 7 d after treatment for phenotypic analysis. Percentage and total counts of CD8 + T cells and the ratio of T conv cells to T reg cells in treated tumors. (C) Percentage of Foxp3+ CD4 + T conv cells. (D) PD-1 and Tim3 expression on CD8 + cells. (E and F) (E) Production of granzyme B in CD8 + T cells and (F) MFI of pSTAT1 on T conv , CD8 + , and T reg cells as in B. (G) Production of IFNγ in T reg cells as in B after restimulation with PMA and ionomycin as in B. (H) IFNγ measured by ELISA from the TIL of CL24 untreated and 7 d after VV treatment as in B. Mice were implanted with C24 and treated with VV dnTGFβmm and αPD-1 as in . At day 8 after VV treatment, after three doses of αPD-1 were received, tumors were harvested for TIL analysis. (I–N) Representative flow plots and quantifications of (I) IFNγ+ T reg cells, (J) IFNγ + T conv cells, (K) IFNγ + CD8 + cells, (L) Tim3+ PD-1+ T reg cells, (M) TCF1+ T reg cells, and (N) TCF1+ CD8 + cells. Cytokine analysis was performed direct ex vivo with PMA and ionomycin restimulation. (O) Schematic for P and Q. (P) Tumor growth and survival (bottom) of C57BL/6 mice implanted intradermally with bilateral C24 and, when tumors were ∼20 mm 2 , one was treated with a single IT injection of VV dnTGFbmm (injected) at 2.5 × 10 6 PFU/mouse (black arrowhead). Mice were sacrificed when either tumor reached 15 mm in any direction. Starting at 4 d after VV or PBS treatment, mice were given anti-PD-1 or isotype control IP three times a week. (Q) Average growth of H. Data represent two independent experiments. Each point or line represents an individual mouse (A–P). *P < 0.05, **P < 0.01, ****P < 0.0001 by Welch’s T test (B–H), Mantel-Cox test (A and P), unpaired t Test (I–N), or mixed-effects analysis (Q). ns, non-significant. Error bars indicate SEMs.

Techniques Used: Injection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Ex Vivo

Viral delivery of TGFβ inhibition alleviates immunosuppressive T reg cells in resistant tumors. Foxp3-Ametrine or Foxp3-RFP mice implanted intradermally with MEER vvS or MEER vvR were treated with an IT injection of VV ctrl or VV dnTGFβmm at 2.5 × 10 6 PFU/mouse or PBS control. (A–E) Tumors and lymph nodes were harvested 4 (A) or 7 (B–E) d after treatment for phenotypic analysis. (A) Percentage and total counts of T reg cells at day 4 after treatment. (B) Percentage and MFI of Nrp1+ T reg cells at day 7. (C) Percentage of pSTAT1 Ser727 + T reg cells at day 7. (D) MFI of Foxp3 in T reg cells at day 7. (E) Percentage of LAP-TGFβ1+ T reg cells at day 7. (F) Percentage of T conv cells and CD8 + cells 7 d after treatment. (G) Production of TNFα and IFNγ in T conv cells from treated tumors after restimulation with PMA and ionomycin. (H) Percentage of PD-1- and Tim3-expressing CD8 + cells 7 d after treatment. (I) Percentage of TCF1+ in PD-1 and Tim3 CD8 + populations 7 d after treatment, representative plot of PD-1+Tim3− cells. Data represent three (H and I) or four (A–G) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (A–G) or two-way ANOVA (H and I) with Tukey’s multiple comparison test. ns, non-significant. Error bars indicate SEMs.

Figure Legend Snippet: Viral delivery of TGFβ inhibition alleviates immunosuppressive T reg cells in resistant tumors. Foxp3-Ametrine or Foxp3-RFP mice implanted intradermally with MEER vvS or MEER vvR were treated with an IT injection of VV ctrl or VV dnTGFβmm at 2.5 × 10 6 PFU/mouse or PBS control. (A–E) Tumors and lymph nodes were harvested 4 (A) or 7 (B–E) d after treatment for phenotypic analysis. (A) Percentage and total counts of T reg cells at day 4 after treatment. (B) Percentage and MFI of Nrp1+ T reg cells at day 7. (C) Percentage of pSTAT1 Ser727 + T reg cells at day 7. (D) MFI of Foxp3 in T reg cells at day 7. (E) Percentage of LAP-TGFβ1+ T reg cells at day 7. (F) Percentage of T conv cells and CD8 + cells 7 d after treatment. (G) Production of TNFα and IFNγ in T conv cells from treated tumors after restimulation with PMA and ionomycin. (H) Percentage of PD-1- and Tim3-expressing CD8 + cells 7 d after treatment. (I) Percentage of TCF1+ in PD-1 and Tim3 CD8 + populations 7 d after treatment, representative plot of PD-1+Tim3− cells. Data represent three (H and I) or four (A–G) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (A–G) or two-way ANOVA (H and I) with Tukey’s multiple comparison test. ns, non-significant. Error bars indicate SEMs.

Techniques Used: Inhibition, Injection, Control, Expressing, Comparison

Image Search Results

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: T reg cells in VV-resistant tumors have elevated TGFβ and a repressed response to IFNγ. (A) Luminex cytokine analysis of TIF harvested from untreated MEER vvR or MEER vvS implanted in C57BL/6 mice. Three mice per group, three technical repeats per mouse. (B) Active TGFβ 1–3 concentration in the TIF of untreated MEER vvS or MEER vvR tumors as determined by TGFβ reporter assay. (C) TGFβR2 expression on CD8 + , Foxp3− T conv , or Foxp3+ T reg cells in untreated MEER vvR or MEER vvS implanted in Foxp3-reporter mice. Representative histograms from a MEER vvR tumor. 4 repeats, 10 M vvS , 9 M vvR mice. (D) Experimental schema of E–L and Q. Repeated three times. (E–L and Q) Percentage of (E) LAP-TGFβ1+, (F) GARP+, (G) CD103+, (I) TCF1+, (J) CD25 + , (K) CD122+, (L) PD-1+ Tim3+, and percentage and MFI of (H) Nrp1+ and (Q) pSTAT1+ T reg cells by flow cytometry as in D. (M) IFNγ concentration in TIF of MEER vvR and MEER vvS at 7 d after treatment with PBS or VV as in . (N–P) Percentage of pSTAT1 S727 + (N) CD8 + cells, (O) T conv cells, and (P) T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . (R) Representative flow plots and percentage of IFNγ+ T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . Cells were restimulated with PMA/ionomycin direct ex vivo from tumors. M vvS = MEER vvS , M vvR = MEER vvR . Data represent two (A, B, and I–M), three (C, E–H, and Q), or four (N–P and R) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test (A), unpaired T test (B), one-way ANOVA with Sidak’s multiple comparison test paired T test (C, M–P, and R), or paired t test (E–L and Q). ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Luminex, Concentration Assay, Reporter Assay, Expressing, Flow Cytometry, Ex Vivo, Comparison

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: Phenotyping observed in TIL T reg cells is found in dLN and TIL T conv to a lesser extent. (A–I) Quantification of the percentages of dLN T reg cells from paired tumors in Foxp3-Ametrine or Foxp3-RFP mice as in . (A) LAP-TGFβ1+, (B) GARP+, (C) CD103+, (D) Nrp1+, (E) TCF1+, (F) CD25 + , (G) CD122+, (H) PD-1+ Tim3+, and (I) pSTAT1+. (J–Q) Representative flow plots and quantification of T reg phenotyping markers on tumor-infiltrating T conv cells as in . Representative histograms of tumor-infiltrating T conv (Foxp3−) and T reg (Foxp3+) cells are shown for comparison, and quantification axes are scaled for T reg expression. Quantification and flow plots of (J) LAP-TGFβ1+, (K) GARP+, (L) CD103+, (M) Nrp1+, (N) CD25 + , (O) CD122+, (P) Tim3, and PD-1 (Q) pSTAT1+ Tconv cells. (R–U) Representative flow plots and quantification in paired dLN and tumors as in of (R) CD62L and CD44 on T conv cells, (S) CD62L and CD44 on CD8 + cells, (T) TCF1 on T conv cells, and (U) TCF1 on CD8 + cells. CM, central memory CD62L+ CD44 + ; Eff, effector CD62L− CD44 + ; Naïve, CD62L+ CD44 − . Data represent two (R–U) or three (A–Q) independent experiments. Each point represents an individual mouse *P < 0.05, **P < 0.01, ***P < 0.001 by paired T test (A–Q, T, and U) or one way ANOVA with Sidaks multiple comparisons test (R and S). ns, non-significant. Error bars indicate SEMs.

Techniques: Comparison, Expressing

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: TGFβ limits IFNγ signaling and increases T reg cell stability. (A) Immunoblot and densitometry of pSTAT1 Y701 , STAT1, and β-actin in T reg cells sorted from spleen and lymph node of a Foxp3 reporter mouse and treated for 30 min with IFNγ, TGFβ1, or both. (B) Quantification and Cell Trace Violet (CTV) plots of the proliferation of stimulated Thy1.1+ CD4 responder cells in the presence of suppressing T reg cells at the 1:8 T reg cell:responder ratio in an in vitro suppression assay. Percent suppression is normalized to the proliferation index of stimulated CD4 + responder control without T reg cells. T reg cells were sorted from spleen and lymph node of a Foxp3-reporter mouse and then cultured for 3 d in IFNγ, TGFβ, or both. Cells were then sorted again to purify Foxp3+ T reg cells and then co-cultured in the suppression assay with CTV-labeled responder CD4 + cells. (C) Surface Nrp1 expression on sorted T reg cells from spleen and lymph node of a Foxp3-reporter mouse cultured in vitro in varying TGFβ concentrations for 48 h (D–F) An EV control and TGFβ 1 overexpressing (TGFβ OE) line were generated from the MEER vvS line. (D) Tumor growth of C57BL/6 mice implanted intradermally with MEER vvS-EV or MEER vvS-TGFβ OE and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (E) Survival of D. (F) Average tumor growth of MEER vvS-EV and MEER vvS-TGFβ OE as in D. Data represent two (C), four (A and D–F), or five (B) independent experiments; each point or line represents an individual mouse (A–D). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Sidak’s multiple comparison test (A and C), paired T test (B), Mantel-Cox test (E), or mixed effects analysis (F). ns, non-significant. Error bars indicate SEMs. Source data are available for this figure: .

Techniques: Western Blot, In Vitro, Suppression Assay, Control, Cell Culture, Labeling, Expressing, Generated, Injection, Comparison

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: VVdnTGFbmm only affects T reg cell phenotype in the tumor. (A) Western blot for TGFβ in EV control and TGFβ 1 overexpressing (TGFβ OE) MEER vvS lines and MEER vvR and MEER vvS as in . (B) Active TGFβ 1–3 levels measured in TIF of LLC, MC38 (colon adenocarcinoma), B16-F10 (melanoma), and C24 ( Pten –/– Braf V600E melanoma) tumors in C57Bl/6 mice as in . The average TGFβ concentration of MEER vvS (light blue) and MEER vvR (gray) from are overlaid as dotted lines. (C) Growth curve of LLC tumors treated with PBS or VV (black arrowhead) as in . (D–N) Representative flow plots and quantification of dLN (D–K) and T reg phenotyping markers on tumor infiltrating T conv cells (L–N) in Foxp3-Ametrine or Foxp3-RFP mice as in . Quantification of dLN (D) percent Foxp3+ of CD4 + and (E) MFI of Foxp3. Quantification and representative flow plots of (F) Nrp1+, (G) pSTAT1+, and (H) LAP-TGFβ1 on dLN T reg cells. Quantification of dLN (I) percent Foxp3− of CD4 + and CD8 + . Quantification and representative flow plots of (J) TNFα and IFNγ in T conv cells with direct ex vivo PMA/ionomycin stimulation and (K) PD-1 and Tim3 on CD8 + cells in dLN. Quantification and representative flow plots of (L) Nrp1+, (M) pSTAT1+, and (N) LAP-TGFβ1 on TIL T conv cells. Data represent two (A–C) or four (D–N) independent experiments. Each dot or line represents a technical repeat (A) or mouse (B–N). *P < 0.05 by one-way ANOVA with Sidak’s multiple comparisons test. ns, non-significant. Source data are available for this figure: .

Techniques: Western Blot, Control, Concentration Assay, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: C24 melanoma model has a similar phenotype to MEER vvR after VV treatment. (A) Tumor growth (left, middle) and survival (right) of C57BL/6 mice implanted intradermally with C24 and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (B) Foxp3-Ametrine or Fpxp3-RFP reporter mice implanted intradermally with C24 were as in A. Tumors and lymph nodes were harvested 7 d after treatment for phenotypic analysis. Percentage and total counts of CD8 + T cells and the ratio of T conv cells to T reg cells in treated tumors. (C) Percentage of Foxp3+ CD4 + T conv cells. (D) PD-1 and Tim3 expression on CD8 + cells. (E and F) (E) Production of granzyme B in CD8 + T cells and (F) MFI of pSTAT1 on T conv , CD8 + , and T reg cells as in B. (G) Production of IFNγ in T reg cells as in B after restimulation with PMA and ionomycin as in B. (H) IFNγ measured by ELISA from the TIL of CL24 untreated and 7 d after VV treatment as in B. Mice were implanted with C24 and treated with VV dnTGFβmm and αPD-1 as in . At day 8 after VV treatment, after three doses of αPD-1 were received, tumors were harvested for TIL analysis. (I–N) Representative flow plots and quantifications of (I) IFNγ+ T reg cells, (J) IFNγ + T conv cells, (K) IFNγ + CD8 + cells, (L) Tim3+ PD-1+ T reg cells, (M) TCF1+ T reg cells, and (N) TCF1+ CD8 + cells. Cytokine analysis was performed direct ex vivo with PMA and ionomycin restimulation. (O) Schematic for P and Q. (P) Tumor growth and survival (bottom) of C57BL/6 mice implanted intradermally with bilateral C24 and, when tumors were ∼20 mm 2 , one was treated with a single IT injection of VV dnTGFbmm (injected) at 2.5 × 10 6 PFU/mouse (black arrowhead). Mice were sacrificed when either tumor reached 15 mm in any direction. Starting at 4 d after VV or PBS treatment, mice were given anti-PD-1 or isotype control IP three times a week. (Q) Average growth of H. Data represent two independent experiments. Each point or line represents an individual mouse (A–P). *P < 0.05, **P < 0.01, ****P < 0.0001 by Welch’s T test (B–H), Mantel-Cox test (A and P), unpaired t Test (I–N), or mixed-effects analysis (Q). ns, non-significant. Error bars indicate SEMs.

Techniques: Injection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: Viral delivery of TGFβ inhibition alleviates immunosuppressive T reg cells in resistant tumors. Foxp3-Ametrine or Foxp3-RFP mice implanted intradermally with MEER vvS or MEER vvR were treated with an IT injection of VV ctrl or VV dnTGFβmm at 2.5 × 10 6 PFU/mouse or PBS control. (A–E) Tumors and lymph nodes were harvested 4 (A) or 7 (B–E) d after treatment for phenotypic analysis. (A) Percentage and total counts of T reg cells at day 4 after treatment. (B) Percentage and MFI of Nrp1+ T reg cells at day 7. (C) Percentage of pSTAT1 Ser727 + T reg cells at day 7. (D) MFI of Foxp3 in T reg cells at day 7. (E) Percentage of LAP-TGFβ1+ T reg cells at day 7. (F) Percentage of T conv cells and CD8 + cells 7 d after treatment. (G) Production of TNFα and IFNγ in T conv cells from treated tumors after restimulation with PMA and ionomycin. (H) Percentage of PD-1- and Tim3-expressing CD8 + cells 7 d after treatment. (I) Percentage of TCF1+ in PD-1 and Tim3 CD8 + populations 7 d after treatment, representative plot of PD-1+Tim3− cells. Data represent three (H and I) or four (A–G) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (A–G) or two-way ANOVA (H and I) with Tukey’s multiple comparison test. ns, non-significant. Error bars indicate SEMs.

Techniques: Inhibition, Injection, Control, Expressing, Comparison

The dFab-CCR heterodimerization format activates a broad range of cytokine receptors families in primary human T cells. A, Schematic of the main cytokine receptors family members and the viral vector design selected for the generation of the dFab_CCR library. The illustration was created with BioRender.com (RRID:SCR_018361). B, Quantitated proliferation of T cells engineered with either IL2 or IL7 dFab_CCRs, expressed as CTV MFI dilution (left), and fold expansion (right) of RQR8 + CD3 + T cells ( n = 4, one-way ANOVA; ns P > 0.05, ****, P ≤ 0.0001). C, Immunoblot analysis of STAT5 phosphorylation, Y694. Nontransduced T cells were cultured in cytokine starvation for 24 hours. dFab_CCR-IL2 or -IL7 were cytokine starved for 96 hours. GAPDH was used as loading control. Data are representative of three independent experiments. D, Immunoblot analysis of in vitro STAT6 phosphorylation, Y641. Nontransduced and dFab_CCR-IL4 T cells were cultured in cytokine starvation for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. E, Immunoblot analysis of in vitro STAT3 phosphorylation, Y705. Nontransduced, IL9, and IL21 dFab_CCR T cells were cultured in cytokine starvation for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. F, IFNγ secreted by IL2, IL12, IL23, and IL27 dFab_CCR transduced T cells cultured for either 24 or 48 hours in cytokine starvation ( n = 3, one-way ANOVA; ns P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). G, Immunoblot analysis of in vitro STAT1 and STAT3 phosphorylation. Nontransduced, dFab_CCR-IL12, -IL23, and -IL27 T cells were cytokine starved for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. H, IFNγ secreted by IL2 and IL18 dFab_CCR T cells cultured for either 24 or 48 hours in cytokine starvation ( n = 3, one-way ANOVA; ns P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01). I, Immunoblot analysis of in vitro ERK1/2 phosphorylation, T202 and Y204. Nontransduced, IL1, IL18, and IL33 dFab_CCR T cells were cytokine starved for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. J, Quantitated in vitro proliferation of T cells engineered with either -IL3, -IL5 and -GMCSF dFab_CCRs, expressed as CTV MFI dilution (left), and fold expansion (right) of RQR8 + /CD3 + T cells ( n = 4, one-way ANOVA; ns P > 0.05; *, P ≤ 0.05). K, Immunoblot analysis of in vitro STAT3 and STAT5 phosphorylation. Nontransduced T cells were cytokine starved for 24 hours. IL3, IL5, and GMCSF dFab_CCRs T cells were cytokine starved for 96 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. All data are presented as mean ± SEM.

Journal: Cancer Immunology Research

Article Title: Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors

doi: 10.1158/2326-6066.CIR-22-0640

Figure Lengend Snippet: The dFab-CCR heterodimerization format activates a broad range of cytokine receptors families in primary human T cells. A, Schematic of the main cytokine receptors family members and the viral vector design selected for the generation of the dFab_CCR library. The illustration was created with BioRender.com (RRID:SCR_018361). B, Quantitated proliferation of T cells engineered with either IL2 or IL7 dFab_CCRs, expressed as CTV MFI dilution (left), and fold expansion (right) of RQR8 + CD3 + T cells ( n = 4, one-way ANOVA; ns P > 0.05, ****, P ≤ 0.0001). C, Immunoblot analysis of STAT5 phosphorylation, Y694. Nontransduced T cells were cultured in cytokine starvation for 24 hours. dFab_CCR-IL2 or -IL7 were cytokine starved for 96 hours. GAPDH was used as loading control. Data are representative of three independent experiments. D, Immunoblot analysis of in vitro STAT6 phosphorylation, Y641. Nontransduced and dFab_CCR-IL4 T cells were cultured in cytokine starvation for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. E, Immunoblot analysis of in vitro STAT3 phosphorylation, Y705. Nontransduced, IL9, and IL21 dFab_CCR T cells were cultured in cytokine starvation for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. F, IFNγ secreted by IL2, IL12, IL23, and IL27 dFab_CCR transduced T cells cultured for either 24 or 48 hours in cytokine starvation ( n = 3, one-way ANOVA; ns P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). G, Immunoblot analysis of in vitro STAT1 and STAT3 phosphorylation. Nontransduced, dFab_CCR-IL12, -IL23, and -IL27 T cells were cytokine starved for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. H, IFNγ secreted by IL2 and IL18 dFab_CCR T cells cultured for either 24 or 48 hours in cytokine starvation ( n = 3, one-way ANOVA; ns P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01). I, Immunoblot analysis of in vitro ERK1/2 phosphorylation, T202 and Y204. Nontransduced, IL1, IL18, and IL33 dFab_CCR T cells were cytokine starved for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. J, Quantitated in vitro proliferation of T cells engineered with either -IL3, -IL5 and -GMCSF dFab_CCRs, expressed as CTV MFI dilution (left), and fold expansion (right) of RQR8 + /CD3 + T cells ( n = 4, one-way ANOVA; ns P > 0.05; *, P ≤ 0.05). K, Immunoblot analysis of in vitro STAT3 and STAT5 phosphorylation. Nontransduced T cells were cytokine starved for 24 hours. IL3, IL5, and GMCSF dFab_CCRs T cells were cytokine starved for 96 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. All data are presented as mean ± SEM.

Article Snippet: Human IFNγ (BioLegend, 430115), human IL2 (BioLegend, 431815), and mouse IFNγ (BioLegend, 430804) were detected utilizing the indicated BioLegend ElisaMax Kits, following the manufacturer's instructions.

Techniques: Plasmid Preparation, Western Blot, Phospho-proteomics, Cell Culture, Control, In Vitro, Membrane

Influence of BCG viability on mice splenocyte immune response ( A ) Representative density plots showing intracellular IFN-γ, IL2 and TNF-α staining following 12 hr PPD stimulation of mice splenocytes. Four weeks prior, mice were intraperitoneally immunized with VC, MDP1_#2-cKD BCG or saline for the no immunization control group. Red boxes indicate populations of cytokine producing CD4 + T cells from each immunized group. ( B ) Summary bar graph depicting the percentage of CD4 + cytokine + population presented as mean ± SD from 4-6 mice per group. ( C ) Intracellular cytokine MFIs of T cells in each immunized mice group. ( D ) IFN-γ quantification by ELISA following PPD stimulation of immunized mice splenocytes over 7 days. Data ( B ) presented as mean ± SE from 4 to 6 mice. Data ( C and D ) presented as box and whisker plots with max to min displayed. Statistical significance was determined using Kruskal-wallis with Dunn multiple comparison test for ( C ) and ( D ). *P < 0.05 **P < 0.01.

Journal: Scientific Reports

Article Title: Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression

doi: 10.1038/s41598-023-40941-9

Figure Lengend Snippet: Influence of BCG viability on mice splenocyte immune response ( A ) Representative density plots showing intracellular IFN-γ, IL2 and TNF-α staining following 12 hr PPD stimulation of mice splenocytes. Four weeks prior, mice were intraperitoneally immunized with VC, MDP1_#2-cKD BCG or saline for the no immunization control group. Red boxes indicate populations of cytokine producing CD4 + T cells from each immunized group. ( B ) Summary bar graph depicting the percentage of CD4 + cytokine + population presented as mean ± SD from 4-6 mice per group. ( C ) Intracellular cytokine MFIs of T cells in each immunized mice group. ( D ) IFN-γ quantification by ELISA following PPD stimulation of immunized mice splenocytes over 7 days. Data ( B ) presented as mean ± SE from 4 to 6 mice. Data ( C and D ) presented as box and whisker plots with max to min displayed. Statistical significance was determined using Kruskal-wallis with Dunn multiple comparison test for ( C ) and ( D ). *P < 0.05 **P < 0.01.

Article Snippet: Culture supernatants were then harvested and subsequently used to quantify IFN-γ using the mouse IFN-γ ELISA kit (Biolegend, San Diego, CA), following the manufacturer’s instructions.

Techniques: Staining, Saline, Control, Enzyme-linked Immunosorbent Assay, Whisker Assay, Comparison

Summary of results for cell-based functional characterization assays.

Journal: mAbs

Article Title: Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies

doi: 10.1080/19420862.2023.2231129

Figure Lengend Snippet: Summary of results for cell-based functional characterization assays.

Article Snippet: The T cell-secreted IFNγ and IL-2 were measured by the enzyme-linked immunosorbent assay (ELISA) using human IFNγ (Biolegend, 430115) and human IL-2 (Biolegend, 431816) detection kits according to the manufacturer’s protocol.

Techniques: Functional Assay, Activation Assay

a , Endosomes of Hela cells were loaded with AlexaFluor 488-dextran and transfected with either siRNA against Borcs5 or an Arl8b expression plasmid. Representative images showing the repositioning of late endosomes. b , Hela cells were co-transfected with TLR7-HA and siRNA against Borcs5. IL8 gene induction was quantified by qPCR after 2h stimulation with R848. Bar graph shows a representative experiment. Dot plot shows pooled data of N=4; ratio paired t test, two-tailed. Western blots show TLR7-HA expression. C: cleaved, FL: full-length. c , Hela cells were co-transfected with TLR7-HA and Arl8b and treated as in b . Graphs displayed and analyzed as in B. d , Intracellular TNF staining of indicated Hoxb8 macrophage lines with R848 (10 ng/ml), CpG-B (300 nM), LPS (25ng/ml) or Pam3CSK4 (50ng/ml). Representative data of N=5. Western blots show KO validations. e , TNF ELISA of two Borcs5 KO Hoxb8 macrophages lines targeted with two separate guides after 8h stimulation with increasing concentrations of R848. Representative data (n=3) of N=5; unpaired t test, two-tailed. f , Immunoblot of phosphorylated P-p38, P-ERK and IκBα of Hoxb8 macrophages stimulated with R848 for the indicated times. Representative data of N=3. g , Intracellular TNF staining of indicated Raw246.7 macrophage lines with R848 (10 ng/ml), CpG-B (300 nM), Pam3CSK4 (50ng/ml), LPS (25ng/ml), or PolyIC (500ng/ml). Western blots show KO validation and rescue (* unspecific band). Representative data of N=3. h , Representative image of Raw246.7 expressing Borcs5-GreenLantern (GL) stained for GFP and Lamp1. i , IL8 ELISA of Borcs5 knock-down THP1 macrophages after overnight stimulation with increasing doses of R848 (2.5 and 5ng/ml), ssRNA (5µg/ml), and LPS (50ng/ml). Representative data (n=3) of N=2; unpaired t test, two-tailed. Western blot shows knock-down efficiency. ** P < 0.01 and *** P < 0.001.

Journal: bioRxiv

Article Title: Endosome dysfunction leads to gain-of-function TLR7 and human lupus

doi: 10.1101/2023.04.03.535356

Figure Lengend Snippet: a , Endosomes of Hela cells were loaded with AlexaFluor 488-dextran and transfected with either siRNA against Borcs5 or an Arl8b expression plasmid. Representative images showing the repositioning of late endosomes. b , Hela cells were co-transfected with TLR7-HA and siRNA against Borcs5. IL8 gene induction was quantified by qPCR after 2h stimulation with R848. Bar graph shows a representative experiment. Dot plot shows pooled data of N=4; ratio paired t test, two-tailed. Western blots show TLR7-HA expression. C: cleaved, FL: full-length. c , Hela cells were co-transfected with TLR7-HA and Arl8b and treated as in b . Graphs displayed and analyzed as in B. d , Intracellular TNF staining of indicated Hoxb8 macrophage lines with R848 (10 ng/ml), CpG-B (300 nM), LPS (25ng/ml) or Pam3CSK4 (50ng/ml). Representative data of N=5. Western blots show KO validations. e , TNF ELISA of two Borcs5 KO Hoxb8 macrophages lines targeted with two separate guides after 8h stimulation with increasing concentrations of R848. Representative data (n=3) of N=5; unpaired t test, two-tailed. f , Immunoblot of phosphorylated P-p38, P-ERK and IκBα of Hoxb8 macrophages stimulated with R848 for the indicated times. Representative data of N=3. g , Intracellular TNF staining of indicated Raw246.7 macrophage lines with R848 (10 ng/ml), CpG-B (300 nM), Pam3CSK4 (50ng/ml), LPS (25ng/ml), or PolyIC (500ng/ml). Western blots show KO validation and rescue (* unspecific band). Representative data of N=3. h , Representative image of Raw246.7 expressing Borcs5-GreenLantern (GL) stained for GFP and Lamp1. i , IL8 ELISA of Borcs5 knock-down THP1 macrophages after overnight stimulation with increasing doses of R848 (2.5 and 5ng/ml), ssRNA (5µg/ml), and LPS (50ng/ml). Representative data (n=3) of N=2; unpaired t test, two-tailed. Western blot shows knock-down efficiency. ** P < 0.01 and *** P < 0.001.

Article Snippet: Supernatants were analyzed using the BioLegend ELISA MAXTM Deluxe Set Mouse TNF-α kit or Human IL-8 kit according to the manufacturer’s instructions.

Techniques: Transfection, Expressing, Plasmid Preparation, Two Tailed Test, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Knockdown

$a , Quantification of endosome radial distribution across segmented shells. Graphs show the fraction of late endosomes normalized per shell area. Pooled data from N=3, shaded areas indicate the SD. (Total number of cells analyzed: WT: 790, Borcs7 KO: 971); two-way ANOVA comparing variation between genotypes. b , Quantification of the luminal pH of endosomes by loading macrophages with the endosomal pH reporter pHrhodo Green (25µg/ml) for the indicated amounts of time. Bafilomycin A treatment (100nM) served as negative control. Pooled data of N=3. Shaded areas indicate the SD; two-way ANOVA comparing variation between genotypes. c , Lamp1 flow staining of Hoxb8 macrophages. Bar graph shows pooled data from N=3; unpaired t test, two-tailed. d , Representative calibration curve obtained by ratiometric fluorescence microscopy of Raw246.7 macrophages and representative images. e , TNF ELISA assay from cell culture supernatants of WT, Borcs5 KO and Borcs5 rescue Raw246.7 cells seeded on 10µg/ml fibronectin-coated(F) 6-well glass bottom plates or tissue culture plastic(P) treated with indicated amounts of ligands. Representative data of N=2. f , Autophagic flux was determined by incubating Raw macrophages of the indicated genotype with increasing amounts of Bafilomycin A (10, 50, 100nM) for 1h and reading out the accumulation of LC3-II by western. Representative image of N=2.$

Journal: bioRxiv

Article Title: Endosome dysfunction leads to gain-of-function TLR7 and human lupus

doi: 10.1101/2023.04.03.535356

Figure Lengend Snippet: a , Quantification of endosome radial distribution across segmented shells. Graphs show the fraction of late endosomes normalized per shell area. Pooled data from N=3, shaded areas indicate the SD. (Total number of cells analyzed: WT: 790, Borcs7 KO: 971); two-way ANOVA comparing variation between genotypes. b , Quantification of the luminal pH of endosomes by loading macrophages with the endosomal pH reporter pHrhodo Green (25µg/ml) for the indicated amounts of time. Bafilomycin A treatment (100nM) served as negative control. Pooled data of N=3. Shaded areas indicate the SD; two-way ANOVA comparing variation between genotypes. c , Lamp1 flow staining of Hoxb8 macrophages. Bar graph shows pooled data from N=3; unpaired t test, two-tailed. d , Representative calibration curve obtained by ratiometric fluorescence microscopy of Raw246.7 macrophages and representative images. e , TNF ELISA assay from cell culture supernatants of WT, Borcs5 KO and Borcs5 rescue Raw246.7 cells seeded on 10µg/ml fibronectin-coated(F) 6-well glass bottom plates or tissue culture plastic(P) treated with indicated amounts of ligands. Representative data of N=2. f , Autophagic flux was determined by incubating Raw macrophages of the indicated genotype with increasing amounts of Bafilomycin A (10, 50, 100nM) for 1h and reading out the accumulation of LC3-II by western. Representative image of N=2.

Article Snippet: Supernatants were analyzed using the BioLegend ELISA MAXTM Deluxe Set Mouse TNF-α kit or Human IL-8 kit according to the manufacturer’s instructions.

Techniques: Negative Control, Staining, Two Tailed Test, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot

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