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rabbit polyclonal antibodies against pkm2 (Proteintech)

Name: rabbit polyclonal antibodies against pkm2
Brand: Proteintech
Rating: 96 (627 reviews)

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Proteintech rabbit polyclonal antibodies against pkm2
Figure 1. <t>PKM2</t> is inversely correlated with CD8+ T-cell infiltration in ESCC, as demonstrated by in silico analysis of data from the TCGA database Pearson correlation analysis was used to evaluate the relationship between PKM2 mRNA expression and the expression of immune checkpoint molecules, as well as that of tumor-associated lymphocyte markers. Tpm stands for transcripts per million.

Figure 1. <t>PKM2</t> is inversely correlated with CD8+ T-cell infiltration in ESCC, as demonstrated by in silico analysis of data from the TCGA database Pearson correlation analysis was used to evaluate the relationship between PKM2 mRNA expression and the expression of immune checkpoint molecules, as well as that of tumor-associated lymphocyte markers. Tpm stands for transcripts per million.

Rabbit Polyclonal Antibodies Against Pkm2, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 627 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models."

Article Title: Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models.

Journal: Acta biochimica et biophysica Sinica

doi: 10.3724/abbs.2025019

Figure 1. PKM2 is inversely correlated with CD8+ T-cell infiltration in ESCC, as demonstrated by in silico analysis of data from the TCGA database Pearson correlation analysis was used to evaluate the relationship between PKM2 mRNA expression and the expression of immune checkpoint molecules, as well as that of tumor-associated lymphocyte markers. Tpm stands for transcripts per million.

Figure Legend Snippet: Figure 1. PKM2 is inversely correlated with CD8+ T-cell infiltration in ESCC, as demonstrated by in silico analysis of data from the TCGA database Pearson correlation analysis was used to evaluate the relationship between PKM2 mRNA expression and the expression of immune checkpoint molecules, as well as that of tumor-associated lymphocyte markers. Tpm stands for transcripts per million.

Techniques Used: In Silico, Expressing

Figure 2. PKM2 regulates PD-L1 and PD-L2 expression in tumor cells (A) In HeLa cells, compared with controls, PKM2 knockout significantly reduced PD-L1 expression while increasing PD-L2 expression. A similar pattern was observed in the A549 lung cancer cell line, where PKM2 knockout notably increased PD-L2 expression and decreased PD-L1 expression. (B) Additionally, PKM2 down- regulates GATA3 expression in tumor cells. β-Actin served as the internal control. The images shown are representative samples, with band intensity variations quantified via ImageJ software.

Figure Legend Snippet: Figure 2. PKM2 regulates PD-L1 and PD-L2 expression in tumor cells (A) In HeLa cells, compared with controls, PKM2 knockout significantly reduced PD-L1 expression while increasing PD-L2 expression. A similar pattern was observed in the A549 lung cancer cell line, where PKM2 knockout notably increased PD-L2 expression and decreased PD-L1 expression. (B) Additionally, PKM2 down- regulates GATA3 expression in tumor cells. β-Actin served as the internal control. The images shown are representative samples, with band intensity variations quantified via ImageJ software.

Techniques Used: Expressing, Knock-Out, Control, Software

Figure 3. PKM2 knockout (KO) significantly suppressed tumor cell proliferation and metastasis in a HeLa cell xenograft mouse model (A) PKM2 knockout drastically inhibited the proliferation of HeLa cells in nude mice xenografted with both HeLa cells and PKM2-knockout HeLa cells (n = 5 per group, **P < 0.01). (B) Similarly, PKM2 knockout markedly reduced lung and liver metastases in nude mice after tail vein injection of PKM2- knockout HeLa cells (n = 5 per group). Representative hematoxylin‒eosin (H&E)-stained sections are shown. Scale bar: 25 μm. (C) Immunohistochemical analysis of PKM2, GATA3, PD-L1, PD-L2, and Ki-67 expression in tumor lesions from xenografted mice with HeLa and PKM2-knockout HeLa cells. Scale bar: 25 μm. Average optical density (AOD) scores were calculated for each section via ImageJ software. Statistical significance was determined via a two-tailed independent sample t test. *P < 0.05, **P < 0.01, ***P < 0.001 relative to the control. (D) Clonogenic assays were performed on A549 and HeLa cells with PKM2 knockout. Statistical analysis was conducted via a two-tailed independent sample t test. ***P < 0.001, ****P < 0.0001 compared with the control.

Figure Legend Snippet: Figure 3. PKM2 knockout (KO) significantly suppressed tumor cell proliferation and metastasis in a HeLa cell xenograft mouse model (A) PKM2 knockout drastically inhibited the proliferation of HeLa cells in nude mice xenografted with both HeLa cells and PKM2-knockout HeLa cells (n = 5 per group, **P < 0.01). (B) Similarly, PKM2 knockout markedly reduced lung and liver metastases in nude mice after tail vein injection of PKM2- knockout HeLa cells (n = 5 per group). Representative hematoxylin‒eosin (H&E)-stained sections are shown. Scale bar: 25 μm. (C) Immunohistochemical analysis of PKM2, GATA3, PD-L1, PD-L2, and Ki-67 expression in tumor lesions from xenografted mice with HeLa and PKM2-knockout HeLa cells. Scale bar: 25 μm. Average optical density (AOD) scores were calculated for each section via ImageJ software. Statistical significance was determined via a two-tailed independent sample t test. *P < 0.05, **P < 0.01, ***P < 0.001 relative to the control. (D) Clonogenic assays were performed on A549 and HeLa cells with PKM2 knockout. Statistical analysis was conducted via a two-tailed independent sample t test. ***P < 0.001, ****P < 0.0001 compared with the control.

Techniques Used: Knock-Out, Injection, Staining, Immunohistochemical staining, Expressing, Software, Two Tailed Test, Control

Figure 5. GATA3 overexpression (OE) in PKM2-knockout A549 and HeLa cells restores effects similar to those observed with PKM2, enhancing PD-L1 and reducing PD-L2 expression (A) Overexpression of GATA3 in PKM2-knockout A549 and HeLa cells reinstates the original PKM2- induced regulation, where PD-L1 expression is increased and PD-L2 expression is decreased. (B) GATA3 overexpression also restored the migratory capacity of A549 and HeLa cells following PKM2 knockout. (C) Similarly, GATA3 overexpression re-established the invasive properties of A549 and HeLa cells after PKM2 knockout. Statistical analysis was performed via a two-tailed independent sample t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control.

Figure Legend Snippet: Figure 5. GATA3 overexpression (OE) in PKM2-knockout A549 and HeLa cells restores effects similar to those observed with PKM2, enhancing PD-L1 and reducing PD-L2 expression (A) Overexpression of GATA3 in PKM2-knockout A549 and HeLa cells reinstates the original PKM2- induced regulation, where PD-L1 expression is increased and PD-L2 expression is decreased. (B) GATA3 overexpression also restored the migratory capacity of A549 and HeLa cells following PKM2 knockout. (C) Similarly, GATA3 overexpression re-established the invasive properties of A549 and HeLa cells after PKM2 knockout. Statistical analysis was performed via a two-tailed independent sample t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control.

Techniques Used: Over Expression, Knock-Out, Expressing, Two Tailed Test, Control

Figure 6. PKM2 is inversely correlated with CD8+ T-cell infiltration in a xenograft model in C57BL/6N mice (A) Evaluation of lentiviral shRNA- mediated PKM2 knockdown efficiency at the mRNA level via qRT-PCR. (B) Confirmation of the knockdown efficiency via western blot analysis. (C) Detection of variations in PD-L1 and PD-L2 expression in AKR cells following PKM2 knockdown, as demonstrated by western blot analysis. (D) Development of a xenograft mouse model using the C57BL/6N strain. (E) Macroscopic examination of subcutaneous tumor growth in the PKM2-shRNA2 (n = 5) and control (n = 5) groups. (F) Quantitative analysis of tumor weight. (G) Representative immunohistochemistry images and corresponding quantifications showing differences in the expressions of GATA3, PKM2, PD-L1, PD-L2, CD206, and CD8+ T cells in tumor tissues from xenograft C57BL/6 N mice. Scale bar: 25 μm, magnification, 200×. Average optical density (AOD) scores were calculated for each section via ImageJ software. Differences between groups were analyzed via an independent sample t test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Figure Legend Snippet: Figure 6. PKM2 is inversely correlated with CD8+ T-cell infiltration in a xenograft model in C57BL/6N mice (A) Evaluation of lentiviral shRNA- mediated PKM2 knockdown efficiency at the mRNA level via qRT-PCR. (B) Confirmation of the knockdown efficiency via western blot analysis. (C) Detection of variations in PD-L1 and PD-L2 expression in AKR cells following PKM2 knockdown, as demonstrated by western blot analysis. (D) Development of a xenograft mouse model using the C57BL/6N strain. (E) Macroscopic examination of subcutaneous tumor growth in the PKM2-shRNA2 (n = 5) and control (n = 5) groups. (F) Quantitative analysis of tumor weight. (G) Representative immunohistochemistry images and corresponding quantifications showing differences in the expressions of GATA3, PKM2, PD-L1, PD-L2, CD206, and CD8+ T cells in tumor tissues from xenograft C57BL/6 N mice. Scale bar: 25 μm, magnification, 200×. Average optical density (AOD) scores were calculated for each section via ImageJ software. Differences between groups were analyzed via an independent sample t test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Techniques Used: shRNA, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Control, Immunohistochemistry, Software

Figure 7. Schematic diagram highlighting the species-dependent regulation of PKM2 In a human model, PKM2 enhances PD-L1 expression while suppressing PD-L2 expression, facilitating immune evasion through PD-L1 upregulation. Conversely, in a mouse model, PKM2 suppresses both PD-L1 and PD-L2 expression, leading to increased CD8+ T-cell infiltration upon PKM2 knockdown. KO, knockout; KD, knockdown; OE, overexpression. The blue arrow indicates downregulation; the red arrow indicates upregulation.

Figure Legend Snippet: Figure 7. Schematic diagram highlighting the species-dependent regulation of PKM2 In a human model, PKM2 enhances PD-L1 expression while suppressing PD-L2 expression, facilitating immune evasion through PD-L1 upregulation. Conversely, in a mouse model, PKM2 suppresses both PD-L1 and PD-L2 expression, leading to increased CD8+ T-cell infiltration upon PKM2 knockdown. KO, knockout; KD, knockdown; OE, overexpression. The blue arrow indicates downregulation; the red arrow indicates upregulation.

Techniques Used: Expressing, Knockdown, Knock-Out, Over Expression

Image Search Results

Journal: Acta biochimica et biophysica Sinica

Article Title: Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models.

doi: 10.3724/abbs.2025019

Figure Lengend Snippet: Figure 1. PKM2 is inversely correlated with CD8+ T-cell infiltration in ESCC, as demonstrated by in silico analysis of data from the TCGA database Pearson correlation analysis was used to evaluate the relationship between PKM2 mRNA expression and the expression of immune checkpoint molecules, as well as that of tumor-associated lymphocyte markers. Tpm stands for transcripts per million.

Article Snippet: The membranes were incubated with rabbit polyclonal antibodies against PKM2 (15822-1-AP; Proteintech, Wuhan, China), PD-L1 (28076-1-AP), PD-L2 (18251-1-AP), GATA3 (66400-1-Ig), and βactin (66009-1-Ig).

Techniques: In Silico, Expressing

Journal: Acta biochimica et biophysica Sinica

Article Title: Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models.

doi: 10.3724/abbs.2025019

Figure Lengend Snippet: Figure 2. PKM2 regulates PD-L1 and PD-L2 expression in tumor cells (A) In HeLa cells, compared with controls, PKM2 knockout significantly reduced PD-L1 expression while increasing PD-L2 expression. A similar pattern was observed in the A549 lung cancer cell line, where PKM2 knockout notably increased PD-L2 expression and decreased PD-L1 expression. (B) Additionally, PKM2 down- regulates GATA3 expression in tumor cells. β-Actin served as the internal control. The images shown are representative samples, with band intensity variations quantified via ImageJ software.

Techniques: Expressing, Knock-Out, Control, Software

Journal: Acta biochimica et biophysica Sinica

Article Title: Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models.

doi: 10.3724/abbs.2025019

Figure Lengend Snippet: Figure 3. PKM2 knockout (KO) significantly suppressed tumor cell proliferation and metastasis in a HeLa cell xenograft mouse model (A) PKM2 knockout drastically inhibited the proliferation of HeLa cells in nude mice xenografted with both HeLa cells and PKM2-knockout HeLa cells (n = 5 per group, **P < 0.01). (B) Similarly, PKM2 knockout markedly reduced lung and liver metastases in nude mice after tail vein injection of PKM2- knockout HeLa cells (n = 5 per group). Representative hematoxylin‒eosin (H&E)-stained sections are shown. Scale bar: 25 μm. (C) Immunohistochemical analysis of PKM2, GATA3, PD-L1, PD-L2, and Ki-67 expression in tumor lesions from xenografted mice with HeLa and PKM2-knockout HeLa cells. Scale bar: 25 μm. Average optical density (AOD) scores were calculated for each section via ImageJ software. Statistical significance was determined via a two-tailed independent sample t test. *P < 0.05, **P < 0.01, ***P < 0.001 relative to the control. (D) Clonogenic assays were performed on A549 and HeLa cells with PKM2 knockout. Statistical analysis was conducted via a two-tailed independent sample t test. ***P < 0.001, ****P < 0.0001 compared with the control.

Techniques: Knock-Out, Injection, Staining, Immunohistochemical staining, Expressing, Software, Two Tailed Test, Control

Journal: Acta biochimica et biophysica Sinica

Article Title: Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models.

doi: 10.3724/abbs.2025019

Figure Lengend Snippet: Figure 5. GATA3 overexpression (OE) in PKM2-knockout A549 and HeLa cells restores effects similar to those observed with PKM2, enhancing PD-L1 and reducing PD-L2 expression (A) Overexpression of GATA3 in PKM2-knockout A549 and HeLa cells reinstates the original PKM2- induced regulation, where PD-L1 expression is increased and PD-L2 expression is decreased. (B) GATA3 overexpression also restored the migratory capacity of A549 and HeLa cells following PKM2 knockout. (C) Similarly, GATA3 overexpression re-established the invasive properties of A549 and HeLa cells after PKM2 knockout. Statistical analysis was performed via a two-tailed independent sample t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control.

Techniques: Over Expression, Knock-Out, Expressing, Two Tailed Test, Control

Journal: Acta biochimica et biophysica Sinica

Article Title: Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models.

doi: 10.3724/abbs.2025019

Figure Lengend Snippet: Figure 6. PKM2 is inversely correlated with CD8+ T-cell infiltration in a xenograft model in C57BL/6N mice (A) Evaluation of lentiviral shRNA- mediated PKM2 knockdown efficiency at the mRNA level via qRT-PCR. (B) Confirmation of the knockdown efficiency via western blot analysis. (C) Detection of variations in PD-L1 and PD-L2 expression in AKR cells following PKM2 knockdown, as demonstrated by western blot analysis. (D) Development of a xenograft mouse model using the C57BL/6N strain. (E) Macroscopic examination of subcutaneous tumor growth in the PKM2-shRNA2 (n = 5) and control (n = 5) groups. (F) Quantitative analysis of tumor weight. (G) Representative immunohistochemistry images and corresponding quantifications showing differences in the expressions of GATA3, PKM2, PD-L1, PD-L2, CD206, and CD8+ T cells in tumor tissues from xenograft C57BL/6 N mice. Scale bar: 25 μm, magnification, 200×. Average optical density (AOD) scores were calculated for each section via ImageJ software. Differences between groups were analyzed via an independent sample t test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Techniques: shRNA, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Control, Immunohistochemistry, Software

Journal: Acta biochimica et biophysica Sinica

Article Title: Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models.

doi: 10.3724/abbs.2025019

Figure Lengend Snippet: Figure 7. Schematic diagram highlighting the species-dependent regulation of PKM2 In a human model, PKM2 enhances PD-L1 expression while suppressing PD-L2 expression, facilitating immune evasion through PD-L1 upregulation. Conversely, in a mouse model, PKM2 suppresses both PD-L1 and PD-L2 expression, leading to increased CD8+ T-cell infiltration upon PKM2 knockdown. KO, knockout; KD, knockdown; OE, overexpression. The blue arrow indicates downregulation; the red arrow indicates upregulation.

Techniques: Expressing, Knockdown, Knock-Out, Over Expression

PD-L1 is located at the centrosome and Golgi. ( A ) Western blot analysis indicating the differential expression of PD-L1 in fibroblast and epithelial cells. ( B ) NIH3T3 cells stained with PD-L1 (green) antibody and co-stained with centrosome marker, γ-tubulin (red) (top panel), and cilium marker, acetylated-α-tubulin (red) (bottom panel). Centrosome localized PD-L1 is indicated with the white arrow. ( C ) RCTE cells stained with PD-L1 (green) antibody and co-stained with γ-tubulin (red) (top panel) and acetylated-α-tubulin (red) (bottom panel). Cells were serum starved to induce cilia growth. ( D ) RCTE cells stained with PD-L1 (green) antibody and co-stained with Golgi marker, GIANTIN (red). ( E ) Overexpression of GFP-PD-L1 (green) co-stained with Golgi marker, Giantin (red) in RCTE cells. ( F ) Overexpression of GFP-PD-L1 (green) co-stained with Golgi-associated protein Ift20 (red) in NIH3T3 cells. All cells were counterstained with DAPI (blue). Scale bars, 20 μm.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: PD-L1 is located at the centrosome and Golgi. ( A ) Western blot analysis indicating the differential expression of PD-L1 in fibroblast and epithelial cells. ( B ) NIH3T3 cells stained with PD-L1 (green) antibody and co-stained with centrosome marker, γ-tubulin (red) (top panel), and cilium marker, acetylated-α-tubulin (red) (bottom panel). Centrosome localized PD-L1 is indicated with the white arrow. ( C ) RCTE cells stained with PD-L1 (green) antibody and co-stained with γ-tubulin (red) (top panel) and acetylated-α-tubulin (red) (bottom panel). Cells were serum starved to induce cilia growth. ( D ) RCTE cells stained with PD-L1 (green) antibody and co-stained with Golgi marker, GIANTIN (red). ( E ) Overexpression of GFP-PD-L1 (green) co-stained with Golgi marker, Giantin (red) in RCTE cells. ( F ) Overexpression of GFP-PD-L1 (green) co-stained with Golgi-associated protein Ift20 (red) in NIH3T3 cells. All cells were counterstained with DAPI (blue). Scale bars, 20 μm.

Article Snippet: Primary antibodies used in this study are listed as follows: mouse monoclonal antibodies against acetylated α-tubulin (6-11B-1, Sigma, St. Louis, MO, USA, T7451, 1:4000 used for immunofluorescence [IF]), γ-tubulin (GTU-88, Sigma, T5326, 1:1000 used for IF), α-tubulin (DM1A, sc-32293, 1:1000), Ninein (F-7, Santa Cruz, Dallas, TX, USA, sc-390540, 1:300 for IF), Cep164 (E-9, Santa Cruz, sc-515403, 1:300 used for IF), C-nap1 (E-9, Santa Cruz, sc-515403, 1:300 used for IF), Smo (E-5, Santa Cruz, sc-166685, 1:100 used for IF), Gli1 (C-1, Santa Cruz, sc-515751, 1:500 used for WB), Giantin (9B6, Abcam, Waltham, MA, USA, ab37266, 1:500 used for IF), actin (AC-15, Sigma, A1978, 1:3000 used for Western blot [WB]), GFP-tag (B-2, sc-9996, 1:1000 for WB), and Myc (9E10, sc-40, 1:1000 for WB); rabbit polyclonal antibodies against PD-L1 (Proteintech, Rosemont, IL, USA, 17952-1-AP, 1:300 used for IF, and 1:1000 used for WB), Cep135 (Abcam, ab75005, 1:300 used for IF), Ift140 (Proteintech, 17460-1-AP, 1:300 used for IF, and 1:1000 for WB), Ift20 (Proteintech, 13615-1-AP, 1:300 used for IF, and 1:1000 for WB), Rab8a (Proteintech, 55296-1-AP, 1:300 used for IF, and 1:1000 for WB), BBS5 (Proteintech, 14569-1-AP, 1:300 used for IF, and 1:1000 for WB), ARL13B (Proteintech, 17711-1-AP, 1:2000 for IF), GFP-tag (Proteintech, 50430-2-AP, 1:1000 for WB) and Myc-Tag (CST, 71D10, 1:1000 used for WB); goat polyclonal antibody against polycystin 2 (E-20, Santa Cruz, sc-10377, 1:500 for WB).

Techniques: Western Blot, Quantitative Proteomics, Staining, Marker, Over Expression

PD-L1 regulates ciliogenesis in 3T3 cells. ( A ) Representative images of acetylated-α-tubulin (red) co-stained with Arl13B (green) in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Quantitative data of cilium length ( n > 100) in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( C – E ) Representative images of acetylated-α-tubulin (red) and GFP-PD-L1 (green) ( C ), and quantitative data of percentage ciliated cells ( n > 75) ( D ), and cilium length ( n > 75) ( E ), in NIH3T3 cells transfected with GFP-PD-L1. All cells were counterstained with DAPI (blue). The quantitative data was obtained by analyzing only cells that were GFP-positive. Scale bar, 20 μm.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: PD-L1 regulates ciliogenesis in 3T3 cells. ( A ) Representative images of acetylated-α-tubulin (red) co-stained with Arl13B (green) in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Quantitative data of cilium length ( n > 100) in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( C – E ) Representative images of acetylated-α-tubulin (red) and GFP-PD-L1 (green) ( C ), and quantitative data of percentage ciliated cells ( n > 75) ( D ), and cilium length ( n > 75) ( E ), in NIH3T3 cells transfected with GFP-PD-L1. All cells were counterstained with DAPI (blue). The quantitative data was obtained by analyzing only cells that were GFP-positive. Scale bar, 20 μm.

Techniques: Staining, Knockdown, Control, Transfection

PD-L1 affects the Golgi accumulation of Ift20 and ciliary protein trafficking of Rab8a and BBS5. ( A ) NIH3T3 cells co-stained for acetylated-α-tubulin and Ift20 (green) in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Quantitative data of fluorescence intensity of Ift20 in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 75). ( C ) NIH3T3 cells co-stained for acetylated-α-tubulin and the small GTPase Rab8a (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( D ) Quantitative data of Rab8a-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 75). ( E ) NIH3T3 cells co-stained for acetylated-α-tubulin and BBS5 (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( F ) Quantitative data of BBS5-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 60). All cells were counterstained with DAPI (blue). Scale bar, 20 μm.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: PD-L1 affects the Golgi accumulation of Ift20 and ciliary protein trafficking of Rab8a and BBS5. ( A ) NIH3T3 cells co-stained for acetylated-α-tubulin and Ift20 (green) in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Quantitative data of fluorescence intensity of Ift20 in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 75). ( C ) NIH3T3 cells co-stained for acetylated-α-tubulin and the small GTPase Rab8a (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( D ) Quantitative data of Rab8a-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 75). ( E ) NIH3T3 cells co-stained for acetylated-α-tubulin and BBS5 (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( F ) Quantitative data of BBS5-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 60). All cells were counterstained with DAPI (blue). Scale bar, 20 μm.

Techniques: Staining, Knockdown, Control, Fluorescence

PD-L1 affects the ciliary recruitment of PC-2 and Ift140. ( A ) NIH3T3 cells co-stained for acetylated-α-tubulin and the PC-2 (polycystin 2) (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Quantitative data of PC-2-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 80). ( C ) NIH3T3 cells co-stained for acetylated-α-tubulin and the Ift140 (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( D ) Quantitative data of Ift140-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 80). All cells were counterstained with DAPI (blue). Scale bars, 20 μm.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: PD-L1 affects the ciliary recruitment of PC-2 and Ift140. ( A ) NIH3T3 cells co-stained for acetylated-α-tubulin and the PC-2 (polycystin 2) (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Quantitative data of PC-2-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 80). ( C ) NIH3T3 cells co-stained for acetylated-α-tubulin and the Ift140 (green), in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( D ) Quantitative data of Ift140-positive cilia in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 80). All cells were counterstained with DAPI (blue). Scale bars, 20 μm.

Techniques: Staining, Knockdown, Control

PD-L1 regulates PC-2 cilia localization in a BBS5-independent manner. ( A ) Western blot analysis evaluating the protein level of BBS5 after siRNA knockdown in NIH3T3 cells compared to control siRNA cells. ( B ) Representative images of acetylated-α-tubulin (red) co-stained with Arl13B (green) in BBS5 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( C ) Quantitative data of cilium length in BBS5 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 100). ( D ) Representative images of acetylated-α-tubulin (red) co-stained with PC-2 (green) in PD-L1 and BBS5 single knockdown, and PD-L1:BBS5 siRNA double knockdown in 3T3 cells compared to control siRNA cells. ( E ) Quantitative data of PC-2-positive cilia in PD-L1 and BBS5 single knockdown, and PD-L1:BBS5 siRNA double knockdown in 3T3 cells compared to control siRNA cells ( n > 75). All cells were counterstained with DAPI (blue). “ns” implies not significant. Scale bars, 20 μm.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: PD-L1 regulates PC-2 cilia localization in a BBS5-independent manner. ( A ) Western blot analysis evaluating the protein level of BBS5 after siRNA knockdown in NIH3T3 cells compared to control siRNA cells. ( B ) Representative images of acetylated-α-tubulin (red) co-stained with Arl13B (green) in BBS5 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( C ) Quantitative data of cilium length in BBS5 siRNA knockdown NIH3T3 cells compared to control siRNA cells ( n > 100). ( D ) Representative images of acetylated-α-tubulin (red) co-stained with PC-2 (green) in PD-L1 and BBS5 single knockdown, and PD-L1:BBS5 siRNA double knockdown in 3T3 cells compared to control siRNA cells. ( E ) Quantitative data of PC-2-positive cilia in PD-L1 and BBS5 single knockdown, and PD-L1:BBS5 siRNA double knockdown in 3T3 cells compared to control siRNA cells ( n > 75). All cells were counterstained with DAPI (blue). “ns” implies not significant. Scale bars, 20 μm.

Techniques: Western Blot, Knockdown, Control, Staining

PD-L1 interacts with BBS5 and PC-2. ( A ) Western blot analysis of the expression of PC-2, Ift140, BBS5, Rab8a and Ift20 in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Western blot analysis of the expression of PC-2, Ift140, BBS5, Rab8a and Ift20 in GFP-PD-L1 overexpressed NIH3T3 cells compared to GFP-vector control cells. ( C ) Western blot analysis of the expression of PC-2, IFT140, BBS5, RAB8a and IFT20 in PD-L1 siRNA knockdown RCTE cells compared to control siRNA cells. ( D ) Western blot of the co-immunoprecipitation analysis between GFP-PD-L1 and BBS5, and PC-2, in HEK293T cells. ( E ) Western blot of the co-immunoprecipitation analysis between Myc-PC-2 and BBS5 and Myc-PC-2 and GFP-PD-L1 in HEK293T cells. ( F ) Western blot analysis of the expression of PD-L1 and BBS5 in Myc-PC-2 overexpressed HEK293T cells.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: PD-L1 interacts with BBS5 and PC-2. ( A ) Western blot analysis of the expression of PC-2, Ift140, BBS5, Rab8a and Ift20 in PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( B ) Western blot analysis of the expression of PC-2, Ift140, BBS5, Rab8a and Ift20 in GFP-PD-L1 overexpressed NIH3T3 cells compared to GFP-vector control cells. ( C ) Western blot analysis of the expression of PC-2, IFT140, BBS5, RAB8a and IFT20 in PD-L1 siRNA knockdown RCTE cells compared to control siRNA cells. ( D ) Western blot of the co-immunoprecipitation analysis between GFP-PD-L1 and BBS5, and PC-2, in HEK293T cells. ( E ) Western blot of the co-immunoprecipitation analysis between Myc-PC-2 and BBS5 and Myc-PC-2 and GFP-PD-L1 in HEK293T cells. ( F ) Western blot analysis of the expression of PD-L1 and BBS5 in Myc-PC-2 overexpressed HEK293T cells.

Techniques: Western Blot, Expressing, Knockdown, Control, Plasmid Preparation, Immunoprecipitation

PD-L1 regulates Hedgehog signal transduction. ( A ) Representative images of acetylated-α-tubulin (red) co-stained with Gli3 (green), in PD-L1 siRNA knockdown NIH3T3 cells without SAG stimulation, compared to control siRNA cells ( n > 100). All cells were counterstained with DAPI (blue). Scale bars, 20 μm. ( B ) Quantitative data of Gli3-positive cilia in unstimulated PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( C ) Western blot analysis of the protein levels of Gli3 and Gli1 in unstimulated PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( D ) qRT-PCR analysis of Hh signaling mediators (Gli1, Gli2 and Gli3) in unstimulated PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. “ns” implies not significant.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: PD-L1 regulates Hedgehog signal transduction. ( A ) Representative images of acetylated-α-tubulin (red) co-stained with Gli3 (green), in PD-L1 siRNA knockdown NIH3T3 cells without SAG stimulation, compared to control siRNA cells ( n > 100). All cells were counterstained with DAPI (blue). Scale bars, 20 μm. ( B ) Quantitative data of Gli3-positive cilia in unstimulated PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( C ) Western blot analysis of the protein levels of Gli3 and Gli1 in unstimulated PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. ( D ) qRT-PCR analysis of Hh signaling mediators (Gli1, Gli2 and Gli3) in unstimulated PD-L1 siRNA knockdown NIH3T3 cells compared to control siRNA cells. “ns” implies not significant.

Techniques: Transduction, Staining, Knockdown, Control, Western Blot, Quantitative RT-PCR

Working model for PD-L1 in the regulation of ciliogenesis. Enriched expression of receptors on the ciliary membrane makes the primary cilium a specialized organelle for receiving and transducing extracellular stimuli into cells. Vesicles carrying ciliary proteins leave the Golgi and move toward the basal body of the primary cilium. Active forms of Rab8, a master modulator for the ciliary protein trafficking, and the BBSome complex, regulates the entry of protein cargo to the cilium. The activities and basal body localization of Rab8 are modulated by Golgi-associated Ift20 and the BBSome. Knockdown of PD-L1 enhances ciliogenesis by modulating the localization of proteins important for ciliary protein sorting, trafficking, and cilia sensory signaling. Depletion of PD-L1 increases cilia length and increases the ciliary recruitment of Rab8a and BBS5. Also, knockdown of PD-L1 increases the ciliary localization of cilia sensory receptor PC-2, and Gli3 which results in the repression of the Hh signaling pathway.

Journal: Cells

Article Title: The Immune Checkpoint Protein PD-L1 Regulates Ciliogenesis and Hedgehog Signaling

doi: 10.3390/cells13121003

Figure Lengend Snippet: Working model for PD-L1 in the regulation of ciliogenesis. Enriched expression of receptors on the ciliary membrane makes the primary cilium a specialized organelle for receiving and transducing extracellular stimuli into cells. Vesicles carrying ciliary proteins leave the Golgi and move toward the basal body of the primary cilium. Active forms of Rab8, a master modulator for the ciliary protein trafficking, and the BBSome complex, regulates the entry of protein cargo to the cilium. The activities and basal body localization of Rab8 are modulated by Golgi-associated Ift20 and the BBSome. Knockdown of PD-L1 enhances ciliogenesis by modulating the localization of proteins important for ciliary protein sorting, trafficking, and cilia sensory signaling. Depletion of PD-L1 increases cilia length and increases the ciliary recruitment of Rab8a and BBS5. Also, knockdown of PD-L1 increases the ciliary localization of cilia sensory receptor PC-2, and Gli3 which results in the repression of the Hh signaling pathway.

Techniques: Expressing, Membrane, Knockdown

(a) ELISA analysis showing endogenous expression of PD-L1 in non-malignant tissues of naïve mice and melanoma tissue removed from a mouse hindpaw 4 w after melanoma cell inoculation. Note that PD-L1 is widely expressed in various non-malignant tissues. n = 3 mice/group. (b) Inhibition of endogenous PD-L1 and PD-1 induces mechanical allodynia in naïve mice. PD-L1 was neutralized with soluble PD-1 (sPD-1, 5 µg, i.pl.), and PD-1 was blocked by monoclonal antibodies RMP1-14 (mouse anti-PD-1 antibody, 5 µg, i.pl.) and Nivolumab (human anti-PD-1 antibody, 10 µg, i.pl.). *P<0.05, vs. human IgG, repeated measures Two-Way ANOVA, n = 5 mice/group. Arrow indicates drug injection. (c,d) Reduced mechanical and thermal pain threshold in Pd1−/− mice, as shown in von Frey test (c) and hot plate test (d). *P<0.05, Two-tailed student t-test, n = 6 mice/group. Data are mean ± s.e.m.

Journal: Nature neuroscience

Article Title: PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1

doi: 10.1038/nn.4571

Figure Lengend Snippet: (a) ELISA analysis showing endogenous expression of PD-L1 in non-malignant tissues of naïve mice and melanoma tissue removed from a mouse hindpaw 4 w after melanoma cell inoculation. Note that PD-L1 is widely expressed in various non-malignant tissues. n = 3 mice/group. (b) Inhibition of endogenous PD-L1 and PD-1 induces mechanical allodynia in naïve mice. PD-L1 was neutralized with soluble PD-1 (sPD-1, 5 µg, i.pl.), and PD-1 was blocked by monoclonal antibodies RMP1-14 (mouse anti-PD-1 antibody, 5 µg, i.pl.) and Nivolumab (human anti-PD-1 antibody, 10 µg, i.pl.). *P<0.05, vs. human IgG, repeated measures Two-Way ANOVA, n = 5 mice/group. Arrow indicates drug injection. (c,d) Reduced mechanical and thermal pain threshold in Pd1−/− mice, as shown in von Frey test (c) and hot plate test (d). *P<0.05, Two-tailed student t-test, n = 6 mice/group. Data are mean ± s.e.m.

Article Snippet: After the transfer, the blots were incubated overnight at 4°C with polyclonal antibody against PD-1 (1:1000, rabbit; Sigma, Catalog: PRS4065).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Inhibition, Injection, Hot Plate Test, Two Tailed Test

(a–d) In situ hybridization (ISH) images showing Pd1 mRNA expression in DRG of wild-type (WT) not Pd1 knockout (Pd1−/−) mice. (a) Low magnification image of ISH with anti-sense probe showing Pd1 mRNA in DRG neurons of WT mice. Scale, 50 µm. (b) High magnification image of double ISH (red) and Nissl staining (green) in DRG sections. Scale, 20 µm. (c) ISH image showing loss of Pd1 mRNA expression in DRG neurons in Pd1−/− mice. Scale, 50 µm. (d) ISH image of sense control probe. Scale, 50 µm. (e) Left, image of immunostaining showing broad PD-1 expression in mouse DRG neurons. Middle, PD-1 expression is lost in Pd1−/− mice. Right, absence of PD-1 immunostaining by the treatment of a blocking peptide. Blue DAPI staining shows all the cell nuclei in DRG sections. Scale, 50 µm. (f) Size frequency distribution of PD-1-positive and total neurons in mouse DRGs. A total of 1555 neurons from 4 WT mice were analyzed. (g,h) Double staining of PD-1 and NF200 in DRG (g) and sciatic nerve (h) sections of mice. Note that PD-1 expression in both NF200-positive and NF200-negative DRG neurons and sciatic nerve axons. Scales, 50 µm. (i) Double immunostaining of PD-1 and CGRP in mouse sciatic nerve. PD-1 is present in axons co-expressing CGRP. Scale, 50 µm. Arrows in g–i indicate the double-labeled neurons and axons.

Journal: Nature neuroscience

Article Title: PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1

doi: 10.1038/nn.4571

Figure Lengend Snippet: (a–d) In situ hybridization (ISH) images showing Pd1 mRNA expression in DRG of wild-type (WT) not Pd1 knockout (Pd1−/−) mice. (a) Low magnification image of ISH with anti-sense probe showing Pd1 mRNA in DRG neurons of WT mice. Scale, 50 µm. (b) High magnification image of double ISH (red) and Nissl staining (green) in DRG sections. Scale, 20 µm. (c) ISH image showing loss of Pd1 mRNA expression in DRG neurons in Pd1−/− mice. Scale, 50 µm. (d) ISH image of sense control probe. Scale, 50 µm. (e) Left, image of immunostaining showing broad PD-1 expression in mouse DRG neurons. Middle, PD-1 expression is lost in Pd1−/− mice. Right, absence of PD-1 immunostaining by the treatment of a blocking peptide. Blue DAPI staining shows all the cell nuclei in DRG sections. Scale, 50 µm. (f) Size frequency distribution of PD-1-positive and total neurons in mouse DRGs. A total of 1555 neurons from 4 WT mice were analyzed. (g,h) Double staining of PD-1 and NF200 in DRG (g) and sciatic nerve (h) sections of mice. Note that PD-1 expression in both NF200-positive and NF200-negative DRG neurons and sciatic nerve axons. Scales, 50 µm. (i) Double immunostaining of PD-1 and CGRP in mouse sciatic nerve. PD-1 is present in axons co-expressing CGRP. Scale, 50 µm. Arrows in g–i indicate the double-labeled neurons and axons.

Article Snippet: After the transfer, the blots were incubated overnight at 4°C with polyclonal antibody against PD-1 (1:1000, rabbit; Sigma, Catalog: PRS4065).

Techniques: In Situ Hybridization, Expressing, Knock-Out, Staining, Immunostaining, Blocking Assay, Double Staining, Double Immunostaining, Labeling

(a) PD-1 immunostaining in a human DRG section. Blue DAPI staining labels all nuclei of cells in DRG. Scale, 50 µm. (b,c) In vitro patch-clamp recording in dissociated small-diameter human DRG neurons (30–50 µm). (b) Suppression of evoked action potential firing by PD-L1. Insert shows a human DRG neuron with a recording pipette. Scale, 25 µm. Blue and red arrows show the shift of RMP after the PD-L1 treatment. (c) Percentage change of action potential frequency (left) and rheobase change (right) following PD-L1 perfusion (10 ng/ml). *P<0.05, vs. vehicle, Two-tailed Student’s t-test, n = 7–10 neurons/3 donors. (d) Reduction of RMP after PD-L1 perfusion. Right, quantification of RMP change. *P<0.05, vs. vehicle, Two-tailed Student’s t-test, n = 13 and 17 neurons/3 donors. (e) Inhibited of transient sodium currents in dissociated human DRG neurons by PD-L1 (10 ng/ml) and the effect of SSG (11 µM). Left, traces of sodium currents. Right, time course of relative sodium currents showing time-dependent inhibition by PD-L1. *P<0.05, Two-Way repeated measures ANOVA, n = 5–8 neurons/2 donors. Data are mean ± s.e.m.

Journal: Nature neuroscience

Article Title: PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1

doi: 10.1038/nn.4571

Figure Lengend Snippet: (a) PD-1 immunostaining in a human DRG section. Blue DAPI staining labels all nuclei of cells in DRG. Scale, 50 µm. (b,c) In vitro patch-clamp recording in dissociated small-diameter human DRG neurons (30–50 µm). (b) Suppression of evoked action potential firing by PD-L1. Insert shows a human DRG neuron with a recording pipette. Scale, 25 µm. Blue and red arrows show the shift of RMP after the PD-L1 treatment. (c) Percentage change of action potential frequency (left) and rheobase change (right) following PD-L1 perfusion (10 ng/ml). *P<0.05, vs. vehicle, Two-tailed Student’s t-test, n = 7–10 neurons/3 donors. (d) Reduction of RMP after PD-L1 perfusion. Right, quantification of RMP change. *P<0.05, vs. vehicle, Two-tailed Student’s t-test, n = 13 and 17 neurons/3 donors. (e) Inhibited of transient sodium currents in dissociated human DRG neurons by PD-L1 (10 ng/ml) and the effect of SSG (11 µM). Left, traces of sodium currents. Right, time course of relative sodium currents showing time-dependent inhibition by PD-L1. *P<0.05, Two-Way repeated measures ANOVA, n = 5–8 neurons/2 donors. Data are mean ± s.e.m.

Article Snippet: After the transfer, the blots were incubated overnight at 4°C with polyclonal antibody against PD-1 (1:1000, rabbit; Sigma, Catalog: PRS4065).

Techniques: Immunostaining, Staining, In Vitro, Patch Clamp, Transferring, Two Tailed Test, Inhibition

(a) Tumor growth after melanoma cell inoculation (MCI) in a hindpaw. Left, images of ipsilateral hindpaw (red arrow) and contralateral hindpaw and an isolated melanoma (top) at MCI-4w. Scales, 5 mm. Right, time course of tumor growth after MCI, revealed by hindpaw volume change. BL, baseline. *P<0.05, vs. BL, One-Way ANOVA, n = 25 mice/group. (b) Serum PD-L1 levels in sham control mice and melanoma-bearing mice (MCI-4w). *P<0.05, two-tailed Student’s t-test. n= 6 mice/group. (c,d) Time course of mechanical pain (c) and spontaneous pain (duration of licking/flinching, d) after MCI. Note that tumor growth is not associated with the development of mechanical allodynia and spontaneous pain. n = 21 and 25 mice/group. (e) Induction of spontaneous pain by soluble PD-1 (sPD-1) following i.pl. injection at MCI-4w. Note a rapid onset of spontaneous pain by sPD-1 within 30 min. *P<0.05, compared with vehicle, two-tailed Student’s t-test. n= 6 and 7 mic/group. (f) Induction of ongoing pain (CPP) in melanoma-bearing mice by sPD-1 (i.pl.). Left, paradigm for assessing CPP in a two-chamber test. Right, difference in time spent in drug-paired compartment between the pre-conditioning and post-conditioning phases. *P<0.05, two-tailed Student’s t-test, n= 7–8 mice/group. (g,h) Induction of mechanical allodynia (g, n=11 mice/group) and spontaneous pain (h, n=9 mice/group) by peri sciatic injection of PD-1-targeting siRNA (2 µg) but not by control non-targeting siRNA (NT, 2 µg), given at MCI-4w. *P<0.05, repeated measures Two-Way ANOVA (g) and two-tailed Student’s t-test (h). (i,j) Intravenous Nivolumab (3 and 10 mg/kg), given at MCI-4w (indicated with an arrow), induces mechanical allodynia (i, n=4–6 mice/group) and spontaneous pain 3 h after the injection (j, n=6 mice/group). *P < 0.05, compared with control human IgG4, repeated measures Two-Way ANOVA (i) and two-tailed Student’s t-test (j). (k,l) Intravenous Nivolumab (10 mg/kg, MCI-4w) increases spontaneous firing of afferent fibers in the sciatic nerve 3 h after the injection. (k) Traces of discharges in melanoma-bearing mice treated with Nivolumab and human IgG4 control. (l) Number of spikes in 2 hours after the treatment. *P<0.05, two-tailed student’s t-test, n = 5 mice/group. Data are expressed as mean ± s.e.m.

Journal: Nature neuroscience

Article Title: PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1

doi: 10.1038/nn.4571

Figure Lengend Snippet: (a) Tumor growth after melanoma cell inoculation (MCI) in a hindpaw. Left, images of ipsilateral hindpaw (red arrow) and contralateral hindpaw and an isolated melanoma (top) at MCI-4w. Scales, 5 mm. Right, time course of tumor growth after MCI, revealed by hindpaw volume change. BL, baseline. *P<0.05, vs. BL, One-Way ANOVA, n = 25 mice/group. (b) Serum PD-L1 levels in sham control mice and melanoma-bearing mice (MCI-4w). *P<0.05, two-tailed Student’s t-test. n= 6 mice/group. (c,d) Time course of mechanical pain (c) and spontaneous pain (duration of licking/flinching, d) after MCI. Note that tumor growth is not associated with the development of mechanical allodynia and spontaneous pain. n = 21 and 25 mice/group. (e) Induction of spontaneous pain by soluble PD-1 (sPD-1) following i.pl. injection at MCI-4w. Note a rapid onset of spontaneous pain by sPD-1 within 30 min. *P<0.05, compared with vehicle, two-tailed Student’s t-test. n= 6 and 7 mic/group. (f) Induction of ongoing pain (CPP) in melanoma-bearing mice by sPD-1 (i.pl.). Left, paradigm for assessing CPP in a two-chamber test. Right, difference in time spent in drug-paired compartment between the pre-conditioning and post-conditioning phases. *P<0.05, two-tailed Student’s t-test, n= 7–8 mice/group. (g,h) Induction of mechanical allodynia (g, n=11 mice/group) and spontaneous pain (h, n=9 mice/group) by peri sciatic injection of PD-1-targeting siRNA (2 µg) but not by control non-targeting siRNA (NT, 2 µg), given at MCI-4w. *P<0.05, repeated measures Two-Way ANOVA (g) and two-tailed Student’s t-test (h). (i,j) Intravenous Nivolumab (3 and 10 mg/kg), given at MCI-4w (indicated with an arrow), induces mechanical allodynia (i, n=4–6 mice/group) and spontaneous pain 3 h after the injection (j, n=6 mice/group). *P < 0.05, compared with control human IgG4, repeated measures Two-Way ANOVA (i) and two-tailed Student’s t-test (j). (k,l) Intravenous Nivolumab (10 mg/kg, MCI-4w) increases spontaneous firing of afferent fibers in the sciatic nerve 3 h after the injection. (k) Traces of discharges in melanoma-bearing mice treated with Nivolumab and human IgG4 control. (l) Number of spikes in 2 hours after the treatment. *P<0.05, two-tailed student’s t-test, n = 5 mice/group. Data are expressed as mean ± s.e.m.

Article Snippet: After the transfer, the blots were incubated overnight at 4°C with polyclonal antibody against PD-1 (1:1000, rabbit; Sigma, Catalog: PRS4065).

Techniques: Isolation, Two Tailed Test, Injection

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