Journal: bioRxiv

Article Title: Mono-ADP-ribosylation-driven immunosuppression and cross-resistance to therapy through cancer cell intrinsic and extrinsic mechanisms

doi: 10.64898/2026.06.01.729331

Figure Lengend Snippet: (A) qRT-PCR analysis of Parp11 mRNA expression in colon adenocarcinoma cell lines (CT26 and MC38) (n=3) and normal mouse colon tissues (n=15). Data are presented as fold change relative to the normal colon tissues group. (B) qRT-PCR analysis of Parp11 mRNA expression in pancreatic ductal adenocarcinoma cell lines (4662, MH6499c4, and MH6419c5) (n=3) and normal mouse pancreas tissues (n=18). Data are presented as fold change relative to the normal pancreas group. (C) qRT-PCR analysis of Parp11 mRNA levels in MC38 and 4662 cells following treatment with adenosine (100 μM for 4 h). Each group contained three biological replicates. Data are presented as fold change relative to the vehicle group. (D) qRT-PCR analysis of Parp11 mRNA levels in MC38 and 4662 cells following treatment with epinephrine (10 μM for 4 h). Each group contained three biological replicates. Data are presented as fold change relative to the vehicle group. (E) qRT-PCR analysis of Parp11 mRNA levels in MC38 and 4662 cells following treatment with GLP-1 ( – ) (100 nM for 4 h). Each group contained three biological replicates. Data are presented as fold change relative to the vehicle group. Relative mRNA levels were normalized to Gapdh. Each dot represents one biological replicate. Data are presented as mean ± SEM (A–E). Statistical significance was determined using two-tailed unpaired Student’s t test (A–E).

Article Snippet: For stimulation experiments, cells were treated with adenosine (100 μM) (Sigma, CAS A4036), epinephrine (10 μM) (selleckchem, S2521), or GLP-1 ( – ) (100 nM) (Phoenix Pharmaceuticals, 028-13) for 4 h unless otherwise indicated.

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Mono-ADP-ribosylation-driven immunosuppression and cross-resistance to therapy through cancer cell intrinsic and extrinsic mechanisms

doi: 10.64898/2026.06.01.729331

Figure Lengend Snippet: (A) Flow cytometry analysis of the frequency (% of live CD45 + cells) of CD4 + T cells, CD8 + T cells, and NK1.1 + or CD49b + cells in the spleens from C57BL6 or Balb/c mice bearing subcutaneous tumors described in . (B) Gating strategy for immune profiling of tumor and splenic tissues. (C) Flow cytometry analysis of the frequency (% of live CD45 + cells) and numbers of tumor-associated macrophages (TAMs, CD11b + F4/80 + ), polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs, CD11b + Ly6G + ), and monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b + Ly6C + ), in subcutaneous MC38 or CT26 tumors described in and . (D) Flow cytometry analysis of the frequency and number of CD4 + T cells and NK cells in subcutaneous MC38 or CT26 tumors described in and . (E) Flow cytometry analysis of the percentage of IFN-γ + , Granzyme B + , and perforin + OT-1 T cells in the co-culture system described in (n = 3). (F) Flow cytometry analysis of MFI of H2-KbDb on splenocytes isolated from spleens of WT or Parp11 −/− mice (n = 3). (G) Flow cytometry analysis of MFI of H2-KbDb on MC38 cells pretreated with adenosine (100 μM), epinephrine (10 μM), GLP-1 ( – ) (100 nM), or vehicle for 4 h (n = 3). Data are presented as fold change relative to the vehicle group. Each dot represents one biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t test (A, C–G).

Article Snippet: For stimulation experiments, cells were treated with adenosine (100 μM) (Sigma, CAS A4036), epinephrine (10 μM) (selleckchem, S2521), or GLP-1 ( – ) (100 nM) (Phoenix Pharmaceuticals, 028-13) for 4 h unless otherwise indicated.

Techniques: Flow Cytometry, Derivative Assay, Co-Culture Assay, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Mono-ADP-ribosylation-driven immunosuppression and cross-resistance to therapy through cancer cell intrinsic and extrinsic mechanisms

doi: 10.64898/2026.06.01.729331

Figure Lengend Snippet: (A) Flow cytometry analysis of the frequency (% of live CD45 + cells) and the number (per gram of tumor tissue) of CD8 + T cells from subcutaneous MC38 or CT26 tumors overexpressing wild-type PARP11, PARP11 HY mutant, or EV control in C57BL6 (n = 5) or Balb/c (n = 7) mice, as described in and . (B) Flow cytometry analysis of the percentage of IFN-γ + and Granzyme B + CD8 + T cells isolated from subcutaneous MC38 or CT26 tumors overexpressing wild-type PARP11, PARP11 HY mutant, or EV control in C57BL6 (n = 5) or Balb/c (n = 7) mice, as described in and . (C) Killing of MC38-OVA-luc cells pre-treated or not with ITK-7 (1 μM, 48 h) or vehicle, followed by adenosine (100 μM), or epinephrine (10 μM), or GLP-1 (100 nM) before co-culture with OT-1 CTLs for 12 h (OT-1: MC38-OVA-luc = 5:1; n = 3). (D) Killing of MC38-OVA-luc cells or B16F10-OVA-luc cells pretreated with ITK-7 (1 μM, 48 h) or vehicle prior to co-culture with OT-1 CTLs for 12 h (OT-1: MC38-OVA-luc = 5:1; OT-1: B16F10-OVA-luc = 5:1; n = 3). (E) Flow cytometry analysis of mean fluorescence intensity (MFI) of H2-Kd on malignant (CD45 − Podoplanin + ) cells from subcutaneous CT26 tumors overexpressing wild-type PARP11, PARP11 HY mutant, or EV control in Balb/c mice (n = 7), as described in . (F) Flow cytometry analysis of MFI of H2-KbDb on malignant (CD45 − EpCAM + ) cells from subcutaneous MC38 tumors overexpressing wild-type PARP11, PARP11 HY mutant, or EV control in C57BL6 mice (n = 5), as described in . (G) Flow cytometry analysis of MFI of H2-KbDb on malignant (CD45 − EpCAM + ) cells from subcutaneous MC38 tumors expressing sgParp11 or NTC in C57BL6 mice (n = 5), as described in . (H) Flow cytometry analysis of MFI of H2-Kd on malignant (CD45 − PDPN + ) cells from subcutaneous CT26 tumors treated as described in (n = 8). Each dot represents one biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t test (A–H).

Article Snippet: For stimulation experiments, cells were treated with adenosine (100 μM) (Sigma, CAS A4036), epinephrine (10 μM) (selleckchem, S2521), or GLP-1 ( – ) (100 nM) (Phoenix Pharmaceuticals, 028-13) for 4 h unless otherwise indicated.

Techniques: Flow Cytometry, Mutagenesis, Control, Isolation, Co-Culture Assay, Fluorescence, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Mono-ADP-ribosylation-driven immunosuppression and cross-resistance to therapy through cancer cell intrinsic and extrinsic mechanisms

doi: 10.64898/2026.06.01.729331

Figure Lengend Snippet: (A) Flow cytometry analysis of MFI of H2-KbDb on MC38 cells. Tumor cells were pretreated with ITK-7 (1 μM, 48 h) or DMSO, followed by adenosine (100 μM), epinephrine (10 μM), or GLP-1 (100 nM), or vehicle for 4 h prior to analysis (n = 3). Data are presented as fold change relative to the vehicle group. (B) Box plot showing PARP11 mRNA expression levels in 179 human pancreatic adenocarcinoma (PAAD) samples and 171 normal tissue samples from TCGA. *P < 0.05 was considered statistically significant. (C) Flow cytometry analysis of MFI of H2-KbDb on 4662, MH6499c4, and MH6419c5 cells pretreated with ITK-7 (1 μM, 48 h) or DMSO. Data are presented as fold change relative to the control group. (D) Representative tumor images and tumor weights of subcutaneous MH6419c5 tumors. Mice were treated as described in . Each dot represents one biological replicate. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t test for (A), (C) and (D).

Article Snippet: For stimulation experiments, cells were treated with adenosine (100 μM) (Sigma, CAS A4036), epinephrine (10 μM) (selleckchem, S2521), or GLP-1 ( – ) (100 nM) (Phoenix Pharmaceuticals, 028-13) for 4 h unless otherwise indicated.

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Effects of CEE on anxiety-like behaviors and GLP-1R expression in mice. (A) Representative tracking plot from the OFT. (B) Number of entries in the center area (**p = 0.0015. n = 9 mice per group). (C) Time spent in the center area (**p = 0.0056. n = 9 mice per group). (D) Representative track plot of the EPM test. (E) Number of entries in the open arms (**p = 0.0070. n = 9 mice per group). (F) Time spent in the open arms during the EPM test (**p = 0.0056. n = 9 mice per group). (G,H) Protein expression of GLP-1R was detected by Western blot analysis (***p = 0.0002. n = 6 mice per group). (I) Representative images of Immunofluorescence staining of GLP-1R and NeuN with DAPI nuclear counterstaining in the PFC. *p = 0.0113. n = 3 mice per group). NeuN (green) was used to label mature neurons, and DAPI (blue) was used as a nuclear counterstain. GLP-1R (red) was predominantly co-localized with NeuN-positive neurons, indicating neuronal expression. Scale bar = 100 μm. (J) PFC dissection: bregma +1.69 mm. All data are presented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CEE, Chronic ethanol exposure; GLP-1R, Glucagon-like Peptide-1 receptor; OFT, Open Field Test; EPM, Elevated Plus Maze.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Dissection

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Effects of Semaglutide on CEE-induced anxiety-like behaviors and GLP-1R expression. (A) Representative tracking plot from the OFT. (B) Number of entries in the center area (**p < 0.01. n = 19 mice per group). (C) Time spent in the center area (***p < 0.001. n = 19 mice per group). (D) Representative track plot of the EPM test. (E) Number of entries in the open arms (***p < 0.001. n = 19 mice per group). (F) Time spent in the open arms during the EPM test (*p < 0.05, ***p < 0.001. n = 19 mice per group). (G,H) Protein expression of GLP-1R was detected by Western blot analysis (**p < 0.01. n = 6 mice per group). (I) Representative confocal images of Immunofluorescence staining of GLP-1R and NeuN with DAPI nuclear counterstaining in the PFC. (*p < 0.05, **p < 0.01. n = 4 mice per group). NeuN (green) was used to label mature neurons, and DAPI (blue) was used as a nuclear counterstain. GLP-1R (red) was predominantly co-localized with NeuN-positive neurons, indicating neuronal expression. Scale bar = 100 μm. (J) PFC dissection: bregma +1.69 mm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: CEE, Chronic ethanol exposure; GLP-1R, Glucagon-like Peptide-1 receptor; OFT, Open Field Test; EPM, Elevated Plus Maze; Sema, Semaglutide.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Dissection

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Effects of Semaglutide on mitochondrial damage in CEE mice. (A,B) Protein expression of mitochondrial GLP-1R was detected (*p < 0.05, ***p < 0.001. n = 6 mice per group). (C,D) Protein expression of mitochondrial CREB was detected by Western blot analysis (*p < 0.05, **p < 0.01. n = 6 mice per group). (E-J) Protein expression of mitochondrial OXPHOS was detected by Western blot analysis, including complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), complex IV (MTCO1), and complex V (ATP5A) (*p < 0.05, **p < 0.01. n = 6 mice per group). (K) Mitochondrial morphology of neurons in PFC of different groups. Scale bar = 2 μm. (L) Ratio of damaged mitochondria (Representative TEM images were used to calculate the percentage of damaged mitochondria. Mitochondrial damage was defined as the presence of mitochondrial swelling and/or cristae disruption, as described in the Materials and Methods.) (**p < 0.01, ***p < 0.001. n = 5 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: GLP-1R, Glucagon-like Peptide-1 receptor; CREB, cAMP Response Element-Binding; OXPHOS, Oxidative phosphorylation; Sema, Semaglutide; NDUFB8, Ubiquinone oxidoreductase subunit B8; SDHB, Succinate dehydrogenase complex iron sulfur subunit B; UQCRC2, Ubiquinol-cytochrome c reductase core protein 2; MTCO1, Mitochondrially encoded cytochrome c oxidase I; vATP5A, ATP synthase F1 subunit alpha.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Expressing, Western Blot, Disruption, Binding Assay, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Regulation of mitochondrial quality control by Semaglutide in CEE mice. (A) Mitochondrial quality control, including fission, fusion, and mitophagy. (B–D) Protein expression of mitochondrial FIS1, DRP1 and p-DRP1 were detected (*p < 0.05, **p < 0.01, ***p < 0.001. n = 6 mice per group). (E–G) Protein expression of mitochondrial MFN1 and MFN2 were detected (*p < 0.05. n = 6 mice per group). (H–J) Protein expressions of mitochondrial Parkin and Pink1 were detected *p < 0.05, ***p < 0.001. n = 6 mice per group). (K-M) Protein expressions of LC3B and P62 were detected (*p < 0.05, **p < 0.01. n = 6 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: GLP-1R, Glucagon-like Peptide-1 receptor; Sema, Semaglutide; DRP1DRP1, Dynamin-related protein-1; FIS1, Fission, mitochondrial 1; MFN1, Mitofusin 1; MFN2, Mitofusin 2; Pink1, PTEN-induced kinase 1; Parkin, Parkin RBR E3 ubiquitin-protein ligase; LC3, Microtubule-associated protein 1A/1B-light chain 3.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Control, Expressing, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Activation of GLP-1R ameliorates alcohol withdrawal induced anxiety-like behavior by regulating neuronal mitochondrial quality control

doi: 10.3389/fphar.2026.1820128

Figure Lengend Snippet: Synaptic morphology in PFC pyramidal neurons of CEE mice treated with Semaglutide. (A-C) Protein expressions of PSD95 and SYN were detected (*p < 0.05, ***p < 0.001. n = 6 mice per group). (D) Representative images of neurons in PFC labeled using Golgi staining. Scale bar = 20 μm. (E) Sholl analysis of PFC neurons revealing alterations in basal dendritic intersections at distinct distances from the soma. Two-way ANOVA with repeated measures revealed a significant effect of drug treatment and distance from the soma (*p < 0.05, **p < 0.01, ***p < 0.001. n = 4 mice per group). (F,G) Total dendritic length and dendritic branch number (**p < 0.01. n = 4 mice per group). (H) Representative images of dendritic spines by Golgi staining. (I) Spine density. Scale bar = 10 μm. (***P < 0.001. n = 4 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: GLP-1R, Glucagon-like Peptide-1 receptor; Sema, Semaglutide; PSD95, Postsynaptic density protein 95; SYN, Synuclein.

Article Snippet: Immunofluorescence staining was performed using primary antibodies against GLP-1R ( GB113881 , 1:5000, Servicebio) and NeuN (GB11138, 1:5000, Servicebio).

Techniques: Labeling, Staining