Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, or PMA on LIF-induced activation of signal transduction and activation of transcription_(STAT3) and ERK1/2 . Western blot was performed on NHBE cells that had been preincubated with or without AG-490, PD-98059, or PMA and then stimulated with LIF. (a) LIF induced activation of tyrosine phosphorylation of STAT3, and tyrosine phosphorylation of STAT3 was inhibited by AG-490, but not by PD-98059, and not affected by PMA. (b) LIF did not enhance the expression of total-STAT3, and its expression was not affected by AG-490, PD-98059, and PMA. (c) LIF induced activation of phosphorylation of ERK1/2, and ERK1/2 activation was inhibited by PD-98059, but not by AG-490; PMA increased the expression of p-ERK1/2 in NHBE cells, but there were no significant differences between the cells stimulated with LIF and the cells stimulated with LIF in the presence of PMA. (d) and (e) LIF did not enhance the expression of total-ERK1/2, furthermore, AG-490, PD-98059, and PMA also did not affect it. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (target/GAPDH) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Activation Assay, Transduction, Western Blot, Expressing

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by immunocytochemistry_(SABC × 200). Immunocytochemistry was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF ((a) control, (b) PD-98059, (c) AG-490, (d) LIF, (e) PMA, (f) LIF + PD-95059, (g) LIF + PMA, (h) LIF + AG-490, (i) LIF + control siRNA, (j) LIF + sham plasmid, (k) LIF + siRNA-1 against STAT3). LIF induced expression of NK-1R, which was inhibited by AG-490, PD-98059, and siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (positive cells number/total cells number) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Immunocytochemistry, Plasmid Preparation

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by RT-PCR . RT-PCR was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF. (a) LIF induced expression of NK-1R mRNA, and that was inhibited by AG-490 and PD-98059; PMA increased the expression of NK-1R mRNA in NHBE cells. (b) LIF-induced expression of NK-1R mRNA was inhibited by siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio_(target/β-actin) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Synthesis of anti-CTLA4-IR700 and cell-killing specificity of CTLA4-targeted NIR-PIT. A) Evaluation of anti-CTLA4-IR700 by SDS-PAGE (left: Colloidal Blue staining, right: 700 nm fluorescence). Unconjugated anti-CTLA4 antibody was used as a control. B) Evaluation of anti-CTLA4-IR700 by SEC. The APC demonstrated light absorption at 280 and 689 nm and corresponding fluorescence at 700nm. C) Flow-cytometric analysis of CTLA4 expression on cancer cell lines. Representative histograms are shown. D) PI-stained dead cell % after in vitro NIR-PIT on cancer cell lines (n = 4; unpaired t test; ns, not significant). E) Flow-cytometric analysis of CTLA4 expression of splenocytes from non-tumor bearing mouse. Total (surface and intracellular) CTLA4 was stained. F) Flow-cytometric analysis of CTLA4 expression in spleen T cells. Representative histograms and RFI are shown (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01, ***P < 0.001, ****P < 0.0001). RFI, Relative fluorescence intensity. G) Treg percentages of T cells after ex vivo NIR-PIT for splenocytes analyzed by flow-cytometry (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01, ****P < 0.0001).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: SDS Page, Staining, Fluorescence, Control, Expressing, In Vitro, Ex Vivo, Flow Cytometry

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: CTLA4 was expressed by Treg cells more than other T cells in tumor tissue. A) CTLA4 expression in T lymphocyte populations in MC38-luc, LL/2-luc, and MOC2-luc tumors were analyzed by flow cytometry. Representative histograms for surface or total (surface + intracellular) CTLA4 expression in CD8+ T cells (CD3+CD8+), CD4+Fopx3−T cells (CD3+CD4+Foxp3−), and CD4+Foxp3+ Tregs (CD3+CD4+Foxp3+) and relative fluorescence intensity (RFI) of CTLA4 are shown (n = 4; one-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ****P < 0.0001). B) CTLA4hi percentage was calculated based on the total expression. The CTLA4hi gates are shown in histograms in A (n = 4; one-way ANOVA followed by Tukey’s test; ****P < 0.0001).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Selective CTLA4hi cell depletion by CTLA4-targeted NIR-PIT. CTLA4hi cells within MC38-luc tumor, tumor draining lymph node, and spleen were assessed with flow cytometry 3 hours after the light exposure. CTLA4 was stained with anti-CTLA4 (clone UC10-4B9) after fixation and permeabilization. A) Representative dot plots to show CTLA4 expression in live cells, and CTLA4 and CD3 expressions among CD45+ live cells. B) The percentages of CTLA4hi cells among total live cells, CD45+CD3+ T cells and CD45+CD3− non-T hematopoietic cells (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Flow Cytometry, Staining, Expressing

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Efficacy of in vivo CTLA4-targeted NIR-PIT. CTLA4-targeted NIR-PIT was performed on MC38-luc, LL/2-luc and MOC2-luc mouse tumor models. A) Treatment schedule. B) Diagram of NIR-light exposure. The red circle indicates where NIR light was irradiated i.e., NIR-light was exposed only to the tumor. C) Fluorescent imaging before and after NIR-PIT in MC38-luc tumor bearing mouse. D) Bioluminescence imaging (BLI) before (day 6) and after (day 8-11) NIR-PIT in MC38-luc tumor bearing mice. E) Luciferase activity calculated from BLI (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; vs. control group). F) Tumor volume curves (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; *P < 0.05, ****P < 0.0001). G) Survival curves (n = 10; log-rank test with Bonferroni correction; **P < 0.01, ***P < 0.001; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: In Vivo, Irradiation, Imaging, Luciferase, Activity Assay, Control

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Selective cell depletion immediately after the CTLA4-targeted NIR-PIT against MC38-luc tumors. T cell populations after the therapy were analyzed by flow cytometry. A) Treg population in tumors and spleens 3 hours after light exposure. The dot plots show the representative examples of CD4 and Foxp3 expressions in the CD3+ T cells. Scatter plots show the percentage of Tregs in CD3+ cells and ratios of non-regulatory/regulatory CD4+ T cells (CD4+Foxp3−/CD4+Foxp3+) (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01; ***, P < 0.001; ns, not significant). B) CD8+ T cell populations in the tumors 3 hours after light exposure. The dot plots show the representative examples of CD8 and IFN-γ expressions in the CD3+ T cells. Scatter plots show the percentage of intra-tumoral CD8+ T cells among total cells and IFN-γ+ cells among CD8+ T cells (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Flow Cytometry

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Immune cell response after the CTLA4-targeted NIR-PIT. A) Early phase activation shows increased percentages of CD69 positive intratumoral CD8+ T cells and NK cells by flow cytometry 1 day after therapy (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05; ns, not significant). B) The expression of activation markers was analyzed in the regional lymph nodes 3 days after therapy. CD69+ and CD25+ percentages among CD8+ T cells were calculated (n = 5-6; one-way ANOVA followed by Tukey’s test; **P < 0.01; ***P < 0.001; ns, not significant). C) Multiplex immunohistochemical staining of tumors 5 days after the therapy. Representative pictures are shown. Scatter plots show the CD8+ cell density within stroma or tumor and the CD8+/CD4+Foxp3+ cell ratio within tumor (n = 3; one-way ANOVA followed by Tukey’s test; *, P < 0.05; **, P < 0.01, ***, P < 0.001; ns, not significant). White dashed line represents tumor border; Scale bar = 100 μm; pCK, pan cytokeratin.

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Activation Assay, Flow Cytometry, Expressing, Multiplex Assay, Immunohistochemical staining, Staining

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: In vivo efficacy of CTLA4-targeted NIR-PIT against MC38-luc tumor in athymic nude mice. A) Treatment regimen. B) Diagram of NIR-light exposure. The red circle indicates where NIR light was irradiated i.e. only to the tumor. C) Fluorescent imaging demonstrates 700-nm fluorescence before and after NIR-PIT in MC38-luc tumor bearing mouse. D) Bioluminescence imaging (BLI) before (day 5) and after (day 7-10) NIR-PIT. E) Luciferase activity calculated from BLI (n = 13; repeated measures two-way ANOVA; ns, not significant). F) Tumor volume curves (n = 13; repeated measures two-way ANOVA; ns, not significant). G) Survival curves (n = 13; log-rank test with Bonferroni correction; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: In Vivo, Irradiation, Imaging, Fluorescence, Luciferase, Activity Assay