Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Schematic diagram showing the establishment of humanized mice (humice) with human immune system reconstituted in NIKO mice. The presence of human CD45 + cells, NK cells, CD4 + and CD8 + T cells in the mice’s peripheral system was validated by flow cytometry. b Primary LM2 tumor size in humice (control, n = 14; Keytruda, n = 14; ENT, n = 12; PX478, n = 14; ENT + Keytruda, n = 16; PX478 + Keytruda, n = 16) and NIKO mice (control, n = 10; ENT + Keytruda, n = 10; PX478 + Keytruda, n = 10), at Day 21 of treatments. c Lung metastasis of humice (control, n = 6; Keytruda, n = 6; ENT, n = 6; PX478, n = 6; ENT + Keytruda, n = 7; PX478 + Keytruda, n = 7) and NIKO mice (control, n = 5; ENT + Keytruda, n = 5; PX478 + Keytruda, n = 5) bearing LM2 tumors at Day 35 assessed by bioluminescence (BLI) measurement. d Representative bioluminescence (BLI) images showing the lung metastasis of humice and NIKO mice. e Flow cytometric analysis of LM2 tumors harvested from humanized mice. IFNγ, TNFα, and granzyme B expression was examined in tumor-infiltrating human CD8 + T cells and NK cells. N = 5 for each group. f Flow cytometry analysis of LM2 tumors harvested from humanized mice. Expressions of human PD-L1 and PD-L2 were examined in total living cells dissociated from LM2 tumors. N = 5 for each group. Quantification data of flow cytometry ( e , f ) are presented as a box and whiskers, with median values and whiskers of minimum and maximum values. Data for b and c were presented as mean ± SD . P values were determined by one-way ( e , f ) or two-way ( b , c ) ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Flow Cytometry, Control, Expressing

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

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.

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: Staining, Knockdown, Control, Transfection

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.

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: Staining, Knockdown, Control, Fluorescence

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.

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: Staining, Knockdown, Control

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.

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, Knockdown, Control, Staining

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.

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, Expressing, Knockdown, Control, Plasmid Preparation, Immunoprecipitation

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.

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

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.

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: Expressing, Membrane, Knockdown

Journal: bioRxiv

Article Title: Neuron-astrocyte Coupling in Lateral Habenula Mediates Depressive-like Behaviors

doi: 10.1101/2024.11.03.621722

Figure Lengend Snippet: Astrocyte-to-Neuron Crosstalk in the LHb (A) Representative image showing viral expression of GCaMP6s in LHb neurons and hM3Dq in LHb astrocytes. Scale bar, 100 μm. (B-C) In vivo fiber photometry recording of LHb neuronal calcium in response to LHb astrocytic hM3Dq activation. Representative raw trace (B) and bar chart (C) showing mean AUC of LHb neuronal calcium signals following an intraperitoneal injection of saline or clozapine. Each circle represents one mouse. (D) Representative images of c-Fos IHC signals following unilateral astrocytic hM3Dq activation in LHb. Note c-Fos signals are only present in injected side. Green, c-Fos; blue, Hoechst. Scale bar, 40 μm. (E) Quantification of total c-Fos + cells in LHb. (F) Representative image of LHb brain slices with viral expression of cOpn5 in astrocytes. Scale bar, 40 μm. (G) Schematic (left) and representative two-photon image (right) illustrating optogenetic activation of cOpn5-expressing astrocytes while recording two-photon images of LHb astrocytic calcium responses. Scale bar: 50 μm. (H) Plots of delta F/F ratio of calcium signals in LHb astrocytes aligned to laser onset. Solid lines indicate mean and shaded areas indicate SEM. (I) Schematic (left) and representative two-photon image (right) illustrating optogenetic activation of cOpn5-expressing astrocytes while recording two-photon images of LHb neuronal calcium responses. Scale bar: 50 μm. (J) Pie chart illustrating percent abundance of excited neurons by astrocytic cOpn5 activation (left, n = 4 slices from 4 mice). Colored outer circle indicates percentage of three types (according to baseline activity) among astrocytic-hM3Dq-excited neurons. Representative raw calcium traces from the three types of astrocytic cOpn5-excited neurons (right). Blue line represents light-on. (K) Bar graph showing effects of ACSF (control), picrotoxin (GABA A receptor antagonist), PPADS and suramin (ATP-R antagonists, ATP receptor antagonists), DPCPX (ADO A 1 R antagonists, adenosine A1 receptor antagonist), SCH58261 (ADO A 2A R antagonist, adenosine A 2A receptor antagonist) and APV and NBQX (iGlu receptor antagonists) on astrocytic cOpn5-evoked neuronal calcium signals in LHb slices. Each circle represents one neuron. (L, M) Illustration of AAVs used for expressing hM3Dq and ADO1.0m (L) or iGluSnFR (M) in LHb astrocytes (left top), fluorescence images (left bottom) and traces (right) illustrating astrocyte-induced adenosine (L) or glutamate (M) release in LHb slice. Scale bar: 100 μm. (Experiments were performed in TTX). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Data are represented as mean ± SEM.

Article Snippet: The following drugs were applied in the bath: Norepinephrine (MCE, HY-13715), Phenylephrine (10 μM, Sigma), γ-Aminobutyric acid (300 μM, Sigma), Serotonin (20 μM, MCE), Dopamine (10 μM, APExBIO), Clozapine N-oxide (CNO, Sigma), Picrotoxin (100 μM; Tocris), PPADS tetrasodium (50 μM; MCE), Suramin hexasodium salt (75 μM ; APExBIO), DPCPX (300 nM; MCE), SCH582661 (100 nM; MCE), APV (50 μM; Sigma), NBQX (10 μM; MCE), Prazosin hydrochloride (10 μM; MCE), Atipamezole hydrochloride (1 μM; MCE), Propranolol hydrochloride (10 μM; MCE), Tetrodotoxin cltrate (TTX; 1 μM; alomone labs).

Techniques: Expressing, In Vivo, Activation Assay, Injection, Saline, Activity Assay, Control, Fluorescence