expression plasmids for flag-ha-bap1  (Blue Heron Biotech)

Journal: Cancer letters

Article Title: BAP1 maintains HIF-dependent interferon beta induction to suppress tumor growth in clear cell renal cell carcinoma.

doi: 10.1016/j.canlet.2022.215885

Figure Lengend Snippet: Fig. 3. BAP1 promotes ISGF3 activity in a deubiquitinase-dependent manner. (A) SDS-solubilized whole cell lysates of UMRC6 cells expressing control (GFP), BAP1-WT, or BAP1-C91G constructs and blotted with the indicated antibodies (left). Quantification of monoubiquitinated H2A/total H2A protein levels (n = 6) in UMRC6 cells expressing the indicated constructs (right). (B) SDS-solubilized whole cell lysates of UMRC2 cells expressing control (GFP), BAP1-WT, or BAP1-C91G constructs and blotted with the indicated antibodies (left). Quantification of monoubiquitinated H2A/total H2A protein levels (n = 6) in UMRC2 cells expressing the indicated constructs (right). (C) SDS-solubilized whole cell lysates of UMRC6 cells (n = 7) expressing the indicated constructs and blotted with the indicated an tibodies. (D) SDS-solubilized whole cell lysates of UMRC2 cells (n = 7) expressing the indicated constructs and blotted with the indicated antibodies. (E) SDS- solubilized whole cell lysates of Ren-02 cells (n = 3) expressing the indicated constructs and blotted with the indicated antibodies.

Article Snippet: The BAP1 expression plasmid was generated by subcloning FlagBAP1 from pDEST-Flag-HA-BAP1 (gifted by Wade Harper, Addgene plasmid 22539) into pLNCX-GFP (gifted by Wei Xu, University of Wisconsin-Madison).

Techniques: Activity Assay, Expressing, Control, Construct

Journal: Cancer letters

Article Title: BAP1 maintains HIF-dependent interferon beta induction to suppress tumor growth in clear cell renal cell carcinoma.

doi: 10.1016/j.canlet.2022.215885

Figure Lengend Snippet: Fig. 4. BAP1 enhances ISGF3 activity through IFN-β. (A–C) RT-qPCR measurement of the indicated transcripts in Ren-02 cells (n = 6) expressing SCR or BAP1 shRNAs. (D-F) RT-qPCR measurement of the indicated transcripts in UMRC6 cells (n = 3) expressing control (GFP), BAP1-WT, or BAP1-C91G constructs. (G-J) RT- qPCR measurement of the indicated transcripts in UMRC6 cells (n = 6) expressing control (GFP) or BAP1 constructs and control (SCR) or IFNB1 shRNAs. †; p = 0.12. (K) SDS-solubilized whole cell lysates of UMRC6 cells (n = 3) expressing GFP or BAP1 constructs and SCR or IFNB1 shRNAs and blotted with the indicated antibodies.

Article Snippet: The BAP1 expression plasmid was generated by subcloning FlagBAP1 from pDEST-Flag-HA-BAP1 (gifted by Wade Harper, Addgene plasmid 22539) into pLNCX-GFP (gifted by Wei Xu, University of Wisconsin-Madison).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Control, Construct

Journal: Cancer letters

Article Title: BAP1 maintains HIF-dependent interferon beta induction to suppress tumor growth in clear cell renal cell carcinoma.

doi: 10.1016/j.canlet.2022.215885

Figure Lengend Snippet: Fig. 5. BAP1 stimulates ISGF3 activity through STING. (A) SDS-solubilized whole cell lysates of UMRC6 cells (n = 3) expressing control (GFP), BAP1-WT, or BAP1-C91G constructs and blotted with the indicated antibodies. (B) Cytoplasmic and nuclear protein fractions extracted from UMRC6 cells (n = 3) expressing GFP, BAP1-WT, or BAP1-C91G constructs and blotted with the indicated antibodies. (C) SDS-solubilized whole cell lysates of Ren-02 cells (n = 3) expressing control (SCR) or BAP1 shRNAs and blotted with the indicated antibodies. (D) RT-qPCR measurement of STING1 transcripts in UMRC6 cells (n = 6) expressing control (GFP), BAP1- WT, or BAP1-C91G constructs. (E) RT-qPCR measurement of STING1 transcripts in UMRC2 cells (n = 4) expressing control (GFP). BAP1-WT, or BAP1-C91G con structs. (F) RT-qPCR measurement of STING1 transcripts in Ren-02 cells (n = 8) expressing control (SCR) or BAP1 shRNAs. (G) EBC-solubitized lysates of primary kidney cells from WT or Bap1fl/fl mice (n = 3) treated with adenovirus-empty or -Cre and blotted with the indicated antibodies. (H) Odds ratio (OR) for association between BAP1 IHC loss and STING IHC loss in the stroma and cancer cells. Analysis was performed for all samples and for samples within each tumor stage and grade. Images below are STING and BAP1 IHC samples assigned the indicated staining intensity scores. (I) SDS-solubilized whole cell lysates of UMRC6 cells (n = 5) expressing GFP, BAP1-WT. or BAP1-C91G treated with H-151 for 24 h and blotted with the indicated antibodies. +, 1 μM; ++, 5 μM.

Article Snippet: The BAP1 expression plasmid was generated by subcloning FlagBAP1 from pDEST-Flag-HA-BAP1 (gifted by Wade Harper, Addgene plasmid 22539) into pLNCX-GFP (gifted by Wei Xu, University of Wisconsin-Madison).

Techniques: Activity Assay, Expressing, Control, Construct, Quantitative RT-PCR, Staining

Journal: Cancer letters

Article Title: BAP1 maintains HIF-dependent interferon beta induction to suppress tumor growth in clear cell renal cell carcinoma.

doi: 10.1016/j.canlet.2022.215885

Figure Lengend Snippet: Fig. 6. BAPI suppression of tumorigenesis was verified in a ccRCC xenograft model. (A) Images of athymic nude mice (n = 12) injected with Ren-02 cells expressing control (SCR) or BAP1 shRNA, with xenografted tumors below. (B) Quantification of tumor weights. Tumors from individual mice are connected by a line. The difference in tumor weights was calculated and compared using the two-tailed Student’s t-test. (C) EBC lysates were harvested from paired tumors in two mice and blotted with the indicated antibodies. (D) H&E and IHC staining of tumor tissue from a representative mouse. (E) Graph of cell proliferation in the indicated Ren- 02 cell lines measured via XTT assay (n = 3). (F) Images of athymic nude mice (n = 10) injected with Ren-02 cells expressing BAP1 shRNA and control (GFP) or BAP1* (shRNA-resistant BAP1), with xenografted tumors below. (G) Quantification of tumor weights. Tumors from individual mice are connected by a line. The difference in tumor weights was calculated and compared using the two-tailed Student’s t-test. (H) EBC lysates were harvested from paired tumors in two mice and blotted with the indicated antibodies. (I) H&E and IHC staining of tumor tissue from a representative mouse. (J) Graph of cell proliferation of the indicated Ren-02 cell lines measured via XTT assay (n = 3).

Article Snippet: The BAP1 expression plasmid was generated by subcloning FlagBAP1 from pDEST-Flag-HA-BAP1 (gifted by Wade Harper, Addgene plasmid 22539) into pLNCX-GFP (gifted by Wei Xu, University of Wisconsin-Madison).

Techniques: Injection, Expressing, Control, shRNA, Two Tailed Test, Immunohistochemistry, XTT Assay

Journal: Cancer letters

Article Title: BAP1 maintains HIF-dependent interferon beta induction to suppress tumor growth in clear cell renal cell carcinoma.

doi: 10.1016/j.canlet.2022.215885

Figure Lengend Snippet: Fig. 7. Genetic activation of ISGF3 blocks tumor growth by BAP1-deficient cancer cells. (A) SDS- solubilized whole cell lysates of Ren-02 cells (n = 4) expressing SCR or BAP1 shRNA and GFP or IRF9- STAT2C (9-2C) constructs and blotted with the indi cated antibodies. For the IRF9 immunoblot, the first row of quantification represents the IRF9-STAT2C fusion protein, while the second row represents endogenous IRF9. (B) Images of athymic nude mice (n = 9) injected with Ren-02 cells expressing BAP1 shRNA and GFP or IRF9-STAT2C (9-2C), with xeno grafted tumors below. (C) Quantification of tumor weights. Tumors from individual mice are connected by a line. The difference in tumor weights was calculated and compared using the two-tailed Stu dent’s t-test. (D) Graph of cell proliferation in the indicated Ren-02 cell lines measured via XTT assay (n = 3).

Article Snippet: The BAP1 expression plasmid was generated by subcloning FlagBAP1 from pDEST-Flag-HA-BAP1 (gifted by Wade Harper, Addgene plasmid 22539) into pLNCX-GFP (gifted by Wei Xu, University of Wisconsin-Madison).

Techniques: Activation Assay, Expressing, shRNA, Construct, Western Blot, Injection, Two Tailed Test, XTT Assay

Journal: Cancer letters

Article Title: BAP1 maintains HIF-dependent interferon beta induction to suppress tumor growth in clear cell renal cell carcinoma.

doi: 10.1016/j.canlet.2022.215885

Figure Lengend Snippet: Fig. 8. A STING agonist increases ISGF3 activity and slows the growth of BAP1-deficient tumors. (A) SDS-solubilized whole cell lysates of Ren-02 cells (n = 2) expressing SCR or BAP1 shRNAs treated 24 h with 100 pg/ml IFN-β or diABZI (+, 1 μM; ++, 5 μM) and blotted with the indicated antibodies. (B) SDS-solubilized whole cell lysates of UMRC6 cells (n = 3) expressing GFP or BAP1, treated 24 h with 1 μM diABZI, and blotted with the indicated antibodies. (C) Images of representative athymic nude mice (n = 8 per treatment group) bearing Ren-02 shBAP1 tumors and treated twice-weekly with vehicle (control) or 3 mg/kg diABZI via intraperitoneal route (left). Graph of individual Ren-02 shBAP1 tumor volumes relative to size at onset of treatment (right). (D) Relative tumor volume of all mice in each treatment group (n = 8) measured twice weekly. (E) EBC lysates were harvested from tumors in four mice and blotted with the indicated antibodies. (F) H&E and IHC staining of tumor tissue from representative mice.

Article Snippet: The BAP1 expression plasmid was generated by subcloning FlagBAP1 from pDEST-Flag-HA-BAP1 (gifted by Wade Harper, Addgene plasmid 22539) into pLNCX-GFP (gifted by Wei Xu, University of Wisconsin-Madison).

Techniques: Activity Assay, Expressing, Control, Immunohistochemistry