Journal: Pathogens

Article Title: HPV and Other Risk Factors Involved in Pharyngeal Neoplasm—Clinical and Morphopathological Correlations in the Southwestern Region of Romania

doi: 10.3390/pathogens12080984

Figure Lengend Snippet: The primary antibody.

Article Snippet: Anti-HPV , Abcam , K1H8 , Citrate, pH 6 , Mouse monoclonal (K1H8) to HPV , 1:50 , HPV-infected cells.

Techniques: Labeling

Journal: Molecular Cancer

Article Title: LINE-1 promotes tumorigenicity and exacerbates tumor progression via stimulating metabolism reprogramming in non-small cell lung cancer

doi: 10.1186/s12943-022-01618-5

Figure Lengend Snippet: L1-FGGY exhibited an oncogenic role dependent on activation of 12-LOX/GPR31 metabolic pathway. A The relative RNA expression values of 5-LOX, 12-LOX and 15-LOX detected in 147 LUSC tumor samples (77 L1-FGGY + vs. 70 L1-FGGY − ) from TJMUCH. B The OS was compared between 5-LOX + and 5-LOX − , 12-LOX + and 12-LOX − , 15-LOX + and 15-LOX − patients respectively grouped according to mRNA levels. C Different resolutions of IHC staining results of the FGGY, 12-LOX, 15-LOX and GPR31 antigen expression in L1-FGGY − and L1-FGGY + LUSC tumor samples. The statistical results of IHC staining were shown at right. D The relative RNA expression of 12-LOX and 15-LOX detected in H520 OV−CTRL and H520 OV− L1−FGGY , as well as in H520 OV− L1−FGGY treated with either ML355 or PD146176. E The secretion value of 12S-HETE and 15S-HETE detected in H520 OV−CTRL , H520 OV− L1−FGGY , and H520 OV− L1−FGGY treated with either ML355 or PD146176. F The proliferation of H520 OV−CTRL , H520 OV− L1−FGGY , H520 OV− L1−FGGY +sh− GPR31 , and H520 OV− L1−FGGY treated with either ML355 or PD146176 was detected using CCK8 method. G Representative images of H520 OV−CTRL , H520 OV− L1−FGGY , H520 OV− L1−FGGY +sh− GPR31 , and H520 OV− L1−FGGY treated with either ML355 or PD146176 in wound healing assays. The statistical results of migration rate were shown at the right of the panel. (H) Representative images of H520 OV−CTRL , H520 OV− L1−FGGY , H520 OV− L1−FGGY +sh− GPR31 , and H520 OV− L1−FGGY treated with either ML355 or PD146176 in trans-well invasion assays. The statistical results of invasion number were shown at right. The data are shown as mean ± SD with plots. * and ** indicate p < 0.05 and p < 0.01, respectively between the groups as indicated

Article Snippet: The following antibodies were used: anti-GPR31 (Abcam, ab75579), anti-ATPase Na + /K + β2 (Bioss, bs-1152R), anti-Wnt3a (Cell signaling Technology (CST), #2391), anti-Wnt5a (CST, #2530), anti-pGSK-3β (phospho Ser9, CST, #5558), anti-JNK (phospho Thr183/Tyr185, CST, #4671), anti-β-catenin (CST, #8480), anti-HA tag (CST, #5017), anti-flag tag (CST, #14,793), anti-USP24 (Proteintech, 13,126–1-AP), anti-FGGY (Abnova, ABN-H00055277), anti-β-Actin (CST, #4967), and anti-GAPDH (Abcam, ab181602).

Techniques: Activation Assay, RNA Expression, Immunohistochemistry, Expressing, Migration

Journal: Molecular Cancer

Article Title: LINE-1 promotes tumorigenicity and exacerbates tumor progression via stimulating metabolism reprogramming in non-small cell lung cancer

doi: 10.1186/s12943-022-01618-5

Figure Lengend Snippet: L1-FGGY activated Wnt signaling pathway in LUSC via accelerating GPR31 deubiquitination and enhancing 12S-HETE/GPR31 interaction. A The pathway scores of different signaling pathways were compared between L1-FGGY + tissues and L1-FGGY − tissues ( n = 12) detected in nCounter® PanCancer IO-360™ Panel. B The relative RNA expression of Wnt3a, Wnt5a, β-catenin and TCF4 detected in 147 LUSC tumor samples from TJMUCH as mentioned above. C The relative RNA expression of Wnt3a, Wnt5a, β-catenin and TCF4 detected in H520 OV−CTRL and H520 OV− L1−FGGY . D The secretion value of Wnt3a and Wnt5a detected in H520 OV−CTRL and H520 OV− L1−FGGY treated with ML355. The cells treated with DMSO were as controls. E Western blot results to detect protein expression of Wnt3a, Wnt5a, β-catenin, and the phosphorylation level of GSK and JNK in H520 OV−CTRL and H520 OV− L1−FGGY treated with either DMSO or ML355 were shown at left. And western blot results to detect key proteins involved in Wnt signaling pathway in H520 OV− L1−FGGY and H520 OV− L1−FGGY +sh− GPR31 were shown at right. F Western blot results to detect expression of GPR31 from whole lysate, fraction of cell membrane and cytosol individually in H520 OV−CTRL and H520 OV− L1−FGGY . G The relative RNA expression of GPR31 detected in H520 OV−CTRL and H520 OV− L1−FGGY . H FGGY is indicated to have an association with USP24 by STRING. I The cell lysates from H520 OV−CTRL and H520 OV− L1−FGGY were used in immunoprecipitation (IP) with flag antibody to detect the ubiquitination level of flag-GPR31. MG132 (10 μM) was added for 6 h before the cells were harvested. J Interaction of FGGY with USP24 (top) and interaction of GPR31 with USP24 (bottom). H520 cells were transfected with the plasmids as indicated. At 24 h post-transfection, the cell lysates were used in IP and immunoblotting (IB) using the antibodies as indicated. K The cell lysates from H520 sh−CTRL and H520 sh− USP24 were used in IP with flag antibody to detect the ubiquitination level of flag-GPR31. MG132 (10 μM) was added for 6 h before the cells were harvested. L Western blot results to detect protein expression of FGGY and USP24 in H520 OV−CTRL and H520 OV− L1−FGGY were shown on top. IP results to detect protein level of USP24 which has an interaction with flag-GPR31 in H520 OV−CTRL and H520 OV− L1−FGGY were shown below

Article Snippet: The following antibodies were used: anti-GPR31 (Abcam, ab75579), anti-ATPase Na + /K + β2 (Bioss, bs-1152R), anti-Wnt3a (Cell signaling Technology (CST), #2391), anti-Wnt5a (CST, #2530), anti-pGSK-3β (phospho Ser9, CST, #5558), anti-JNK (phospho Thr183/Tyr185, CST, #4671), anti-β-catenin (CST, #8480), anti-HA tag (CST, #5017), anti-flag tag (CST, #14,793), anti-USP24 (Proteintech, 13,126–1-AP), anti-FGGY (Abnova, ABN-H00055277), anti-β-Actin (CST, #4967), and anti-GAPDH (Abcam, ab181602).

Techniques: RNA Expression, Western Blot, Expressing, Immunoprecipitation, Transfection

Journal: Molecular Cancer

Article Title: LINE-1 promotes tumorigenicity and exacerbates tumor progression via stimulating metabolism reprogramming in non-small cell lung cancer

doi: 10.1186/s12943-022-01618-5

Figure Lengend Snippet: Reverse transcriptase inhibitor and 12-LOX inhibitor impaired growth of LUSC xenografts and reversed immunosuppressive microenvironment in vivo. A KLN205 OV−CTRL and KLN205 OV− L1−FGGY were inoculated subcutaneously in the DBA2 mice which were subjected to either NVR (50 mg/kg/day) or ML355 (3 mg/kg/day) treatment, or subjected with the two inhibitors simultaneously. Representative images of the forming tumors were shown. B The tumor volume (left panel) and body weight (right panel) of the mice at various time points upon injection. C The qPCR results of genes involved in AA metabolic pathways (left) and qPCR results of genes involved in Wnt signaling (right) were shown as relative expression values in KLN205 OV−CTRL mouse group, KLN205 OV− L1−FGGY mouse group, NVR-treated KLN205 OV− L1−FGGY mice, ML355-treated KLN205 OV− L1−FGGY mice and NVR + ML355-treated KLN205 OV− L1−FGGY mouse group. D Western blot results to detect expression of FGGY, 12-LOX, and GPR31 in different mouse groups we constructed. E The proportions of various immunocytes in different mouse groups using multispectral immuno-fluorescent staining. F Representative images of multispectral immuno-fluorescent staining results

Article Snippet: The following antibodies were used: anti-GPR31 (Abcam, ab75579), anti-ATPase Na + /K + β2 (Bioss, bs-1152R), anti-Wnt3a (Cell signaling Technology (CST), #2391), anti-Wnt5a (CST, #2530), anti-pGSK-3β (phospho Ser9, CST, #5558), anti-JNK (phospho Thr183/Tyr185, CST, #4671), anti-β-catenin (CST, #8480), anti-HA tag (CST, #5017), anti-flag tag (CST, #14,793), anti-USP24 (Proteintech, 13,126–1-AP), anti-FGGY (Abnova, ABN-H00055277), anti-β-Actin (CST, #4967), and anti-GAPDH (Abcam, ab181602).

Techniques: In Vivo, Injection, Expressing, Western Blot, Construct, Staining

Journal: Cancers

Article Title: Repositioning Fenofibrate to Reactivate p53 and Reprogram the Tumor-Immune Microenvironment in HPV+ Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cancers14020282

Figure Lengend Snippet: Inducible HPV16E6E7 knockdown drives a pleotropic anti-cancer effect in HPV16+ SCC cells. ( A ) Design of short hairpin RNA (shRNA) constructs targeting HPV16E6 or HPV16E7. ( B ) Validation of shRNA constructs to target HPV16E6 and HPV16E7, and suppress colony formation. Immunoblot for HPV16E6 and HPV16E7 ( left ). Uncropped immunoblots are available in . Colony formation assay ( right ). Number of colonies as a percentage of control (CTL) count for HPV16E6E7 knockdown (E6E7 KD) induced by shRNA constructs 4 and 5. * p < 0.05; ** p < 0.01. ( C ) Colony formation assay. Number of colonies as a percentage of CTL. ** p < 0.01. ( D ) Tumorsphere formation assay. The number of tumorspheres is shown as a percentage of the CTL count. ** p < 0.01. ( E ) Cell cycle analysis. Black, gray, and white bars indicate the percentage of the total population in each cell cycle phase for CTL and HPV16E6E7 KD cells. ( F ) Apoptosis assay. Percentage of CTL and HPV16E6E7 UMSCC47 cells that are annexin V+ as measured by flow cytometry. * p < 0.05. ( G ) Senenscence assay. The percentage of CTL and HPV16E6E7 UMSCC47 cells that are B-galactosidase positive. ** p < 0.01.

Article Snippet: Subsequently, immunoblot analyses were performed using antibodies specific to HPV16E6 (abcam, Cambridge, MA, USA), HPV16E7 (CERVIMAX, Vienna, Austria), HIF1α (Cell Signaling, Danvers, MA, USA), PD-L1 (abcam), or GADPH (Millipore).

Techniques: Knockdown, shRNA, Construct, Biomarker Discovery, Western Blot, Colony Assay, Control, Tube Formation Assay, Cell Cycle Assay, Apoptosis Assay, Flow Cytometry

Journal: Cells

Article Title: Expression of Proton-Sensitive GPR31, GPR151, TASK1 and TASK3 in Common Skin Tumors

doi: 10.3390/cells11010027

Figure Lengend Snippet: Immunohistochemistry of SCCs. Immunohistochemical staining for GPR31, GPR151, TASK1, and TASK3 in SCC tissue. ( a ) H&E staining, ( b – e ) Immunohistochemical staining. This SCC (patient 10 = slide number 8931) shows strong expression of GPR151 and TASK3. The tumor cells show a weak, positive expression of GPR31 and TASK1. For more stainings of other SCCs, see . Scale bars represent 200 μm. Overview inserts are two times lower in magnification.

Article Snippet: To avoid unspecific antibody binding, proteins were blocked with blocking solution (ZytoChem Plus HRP Kit/Rabbit, Zytomed, Bargteheide, Germany, REF HRP060-Rb) for 10 min. Tissue sections were incubated with the primary rabbit anti-human GPR31 (1:2000 Anti-GPR31 antibody Abcam, Cambridge, UK, ab75579) GPR151 (1:400;Anti-GPCR GPR151 antibody Life Technologies, Waltham, MA, USA Cat.Nr.PA532803), TASK1 (1:666 Anti-TASK1/TASK1 Abcam, Cambridge, UK, Cat.Nr. ab238588;), TASK3 (1:500 Anti-TASK3/Task3 LSBio, Seattle, WA, USA LS_C406505/186347) polyclonal antibodies at 4 °C overnight.

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Expression of Proton-Sensitive GPR31, GPR151, TASK1 and TASK3 in Common Skin Tumors

doi: 10.3390/cells11010027

Figure Lengend Snippet: Immunohistochemistry of BCCs. Immunohistochemical staining for GPR31, GPR151, TASK1, and TASK3 in BCC tissue. ( a ) H&E staining, ( b – e ) immunohistochemical staining. This BCC (patient 2 = slide number 27) shows very low expression of GPR31, GPR151, TASK1 and TASK3. The epidermis serves as a reference with relatively uniform expression. For more stainings of other BCCs, see . Scale bars represent 200 μm. Overview inserts are two times lower in magnification.

Article Snippet: To avoid unspecific antibody binding, proteins were blocked with blocking solution (ZytoChem Plus HRP Kit/Rabbit, Zytomed, Bargteheide, Germany, REF HRP060-Rb) for 10 min. Tissue sections were incubated with the primary rabbit anti-human GPR31 (1:2000 Anti-GPR31 antibody Abcam, Cambridge, UK, ab75579) GPR151 (1:400;Anti-GPCR GPR151 antibody Life Technologies, Waltham, MA, USA Cat.Nr.PA532803), TASK1 (1:666 Anti-TASK1/TASK1 Abcam, Cambridge, UK, Cat.Nr. ab238588;), TASK3 (1:500 Anti-TASK3/Task3 LSBio, Seattle, WA, USA LS_C406505/186347) polyclonal antibodies at 4 °C overnight.

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Expression of Proton-Sensitive GPR31, GPR151, TASK1 and TASK3 in Common Skin Tumors

doi: 10.3390/cells11010027

Figure Lengend Snippet: Immunohistochemistry of NCN. Immunohistochemical staining for GPR31, GPR151, TASK1 and TASK3 in NCN. ( a ) H&E staining, ( b – e ) immunohistochemical staining. This NCN (patient 3 = slide number 1256) shows weak positive expression of GPR31/GPR151. The tumor cells show a weak positive expression of TASK1 and TASK3 in epidermal parts of the tissue and strong positive expression in dermal portions. For more stainings of other NCN, see . Scale bars represent 200 μm. Overview inserts are two times lower in magnification.

Article Snippet: To avoid unspecific antibody binding, proteins were blocked with blocking solution (ZytoChem Plus HRP Kit/Rabbit, Zytomed, Bargteheide, Germany, REF HRP060-Rb) for 10 min. Tissue sections were incubated with the primary rabbit anti-human GPR31 (1:2000 Anti-GPR31 antibody Abcam, Cambridge, UK, ab75579) GPR151 (1:400;Anti-GPCR GPR151 antibody Life Technologies, Waltham, MA, USA Cat.Nr.PA532803), TASK1 (1:666 Anti-TASK1/TASK1 Abcam, Cambridge, UK, Cat.Nr. ab238588;), TASK3 (1:500 Anti-TASK3/Task3 LSBio, Seattle, WA, USA LS_C406505/186347) polyclonal antibodies at 4 °C overnight.

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Expression of Proton-Sensitive GPR31, GPR151, TASK1 and TASK3 in Common Skin Tumors

doi: 10.3390/cells11010027

Figure Lengend Snippet: Immunohistochemistry of MMs. Immunohistochemical staining for GPR31, GPR151, TASK1 and TASK3 in MM. ( a ) H&E staining, ( b – e ) immunohistochemical staining. This MM (patient 13 = slide number 10968) shows strong expression of TASK1 in both epidermal and dermal parts of the tissue. GPR151 and TASK3 stainings show a weak, positive expression in epidermal tumor nests and negative (GPR151), respectively decreasing expression (TASK3) in dermal tumor nests. This is a typical example for the decreasing expression of the G-proteins/KCNKs in deeper tissue sections (marked with *). For more stainings of other MMs, see . Scale bars represent 200 μm. Overview inserts are two times lower in magnification.

Article Snippet: To avoid unspecific antibody binding, proteins were blocked with blocking solution (ZytoChem Plus HRP Kit/Rabbit, Zytomed, Bargteheide, Germany, REF HRP060-Rb) for 10 min. Tissue sections were incubated with the primary rabbit anti-human GPR31 (1:2000 Anti-GPR31 antibody Abcam, Cambridge, UK, ab75579) GPR151 (1:400;Anti-GPCR GPR151 antibody Life Technologies, Waltham, MA, USA Cat.Nr.PA532803), TASK1 (1:666 Anti-TASK1/TASK1 Abcam, Cambridge, UK, Cat.Nr. ab238588;), TASK3 (1:500 Anti-TASK3/Task3 LSBio, Seattle, WA, USA LS_C406505/186347) polyclonal antibodies at 4 °C overnight.

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Expression of Proton-Sensitive GPR31, GPR151, TASK1 and TASK3 in Common Skin Tumors

doi: 10.3390/cells11010027

Figure Lengend Snippet: Summary of immunohistochemical scoring results for GPR31, GPR151, TASK1 and TASK3 on ( a ) SCCs, ( b ) BCCs, ( c ) NCN and ( d ) MMs. ++/green bar: strongly positive staining with >80% of cells positive and/or staining intensity is high; +/blue bar: 20–80% of cells show a weakly positive/partially positive reaction; −/red bar: <20% of cells with weak staining (=negative reaction). NCN and MM are subdivided into epidermal and dermal portions. Numbers in bars represent the occurrence of the particular score. For additional information on the individual scores, see .

Article Snippet: To avoid unspecific antibody binding, proteins were blocked with blocking solution (ZytoChem Plus HRP Kit/Rabbit, Zytomed, Bargteheide, Germany, REF HRP060-Rb) for 10 min. Tissue sections were incubated with the primary rabbit anti-human GPR31 (1:2000 Anti-GPR31 antibody Abcam, Cambridge, UK, ab75579) GPR151 (1:400;Anti-GPCR GPR151 antibody Life Technologies, Waltham, MA, USA Cat.Nr.PA532803), TASK1 (1:666 Anti-TASK1/TASK1 Abcam, Cambridge, UK, Cat.Nr. ab238588;), TASK3 (1:500 Anti-TASK3/Task3 LSBio, Seattle, WA, USA LS_C406505/186347) polyclonal antibodies at 4 °C overnight.

Techniques: Immunohistochemical staining, Staining