Journal: Biology Open

Article Title: Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila

doi: 10.1242/bio.046524

Figure Lengend Snippet: Induction of UPR observed in wing imaginal discs and IPCs with ectopic Hsc70-3 DN expression. (A–B) Expression of Xbp1-GFP generated by ER stress-dependent splicing of xbp1*-GFP mRNA in wing imaginal discs. Phase contrast (A,B) and fluorescence (A′,B′) micrographs of wing imaginal discs. (A,A′) Control wing disc ( Bx>xbp1*-GFP ). (B,B′) Wing disc expressing a dominant-negative form of Hsc70-3 in the wing pouch region (arrow) ( Bx>hsc70-3 DN , xbp1*-GFP ). (C–E) Fluorescence micrograph of wing discs stained with DAPI (white). (C′–E′) Immunostaining of the wing discs with an anti-GRP78 antibody. (D″) Immunostaining of the wing disc with anti-HA antibody. (C,C′) Fluorescence micrograph of a control wing imaginal disc ( Bx-Gal4/+ ). (D–D″) Wing imaginal disc expressing control Hsc70-3 in the wing pouch region of the imaginal disc ( Bx>hsc70-3 ). (E,E′) Wing imaginal disc expressing a dominant-negative form of Hsc70-3 in the same region ( Bx>hsc70-3 DN ). Anti-GRP78 immunostaining is shown in white. Note that more intense immunofluorescence was observed exclusively in areas expressing Hsc70-3 DN , but not the control protein. (A–F) Relative intensity of anti-GRP78 immunostaining in wing imaginal discs. Immunofluorescence signal intensity in each wing imaginal disc with the control Hsc70-3 ( n =31) or Hsc70-3 DN ( n =25) expression was calculated and normalized to the control value, which was set as 1.0 ( Bx-Gal4/+ ) ( n =25; n.s., not significant, P >0.05; *** P <0.001, Student's t -tests). Error bars represent s.e.m. (G–I) Anti-GRP78 immunostaining of IPCs expressing GFPnls in brains from third-instar larvae. (G) Control IPCs ( ilp2>GFPnls ), (H) IPCs expressing the control Hsc70-3 ( ilp2>hsc70-3, GFPnls ), (I) IPCs expressing Hsc70-3 DN ( ilp2>hsc70-3 DN , GFPnls ). Anti-GRP78 immunostaining is colored in red (G–I; white in G′–I′). Nuclei of IPCs visualized by GFPnls expression are colored green (G–I; white in G″–I″). Arrows in H′ and H″ indicate positions of IPC cells. Note that remarkably higher immunostaining signal was observed in IPCs expressing Hsc70-3 DN , but not the control protein. (J) Relative intensities of anti-GRP78 immunostaining in larval IPCs. Immunofluorescence signal intensities in each IPC expressing Hsc70-3 ( n =25) or Hsc70-3 DN ( n =21) were calculated and normalized to the control value of 1.0 ( ilp2>GFPnls ) ( n =21, * P <0.05, *** P <0.001, Student's t -test). Error bars represent s.e.m. Scale bars: (A–E) 100 µm, (G–I) 50 µm.

Article Snippet: The following primary antibodies were used at the dilution described; rabbit anti-β-galactosidase (MP Biomedicals, #55976) at 1:1000, rabbit anti-GRP78 (Bip) (StressMarq Biosciences Inc., Cadboro Bay, Victoria, Canada) that could recognize Hsp70 family proteins including Hsc70-3 in Drosophila at 1:500, rabbit Cleaved Caspase-3 (Asp175) (#9661, Cell Signaling, Danvers, Massachusetts, USA) at 1:200 for larval brain immunostaining and at 1:150 for wing disc immunostaining, rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) (Cell Signaling, antibody #9578) at 1:500, and rabbit anti-phospho-SAPK/JNK (pThr183, pTyr185) (Calbiochem, La Jolla, CA, USA) at 1:200.

Techniques: Expressing, Generated, Fluorescence, Dominant Negative Mutation, Staining, Immunostaining, Immunofluorescence

Journal: Cell death & disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors.

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: Fig. 2 UBE3A promotes PBRM1 degradation in renal cancer cells. a 786-O cells were transfected with indicated plasmids for 24 h. Before harvested for western blotting analysis, cells were treated with or without 20 µM MG132 for 6 h. b 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. c 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. d 786-O cells were infected with indicated shRNAs. After 72 h, cells were treated with cycloheximide (CHX), and cells were collected for western blot analysis at different timepoints. e 786-O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. f 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for western blotting after treatment with MG132 for 8 h. g 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for Western blotting after treatment with MG132 for 8 h. h 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for western blotting after treatment with MG132 for 8 h.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Transfection, Western Blot, Infection

Journal: Cell death & disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors.

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: Fig. 7 A hypothesis model depicted that PKA phosphorylated UBE3A to prevent UBE3A degrading PBRM1. DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 7. Rac1 and -PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or -PIX siRNA, as indicated, and were stimulated with 10 g/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent im- munoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 8. Schema of the molecular association among NOD2, -PIX, Rac1, and Erbin as discussed in the text.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques:

Journal: Cell Reports Medicine

Article Title: Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes

doi: 10.1016/j.xcrm.2021.100422

Figure Lengend Snippet:

Article Snippet: Human interphotoreceptor retinoid-binding protein (IRBP) peptide 161-180 , GenScript , Cat# RP20268.

Techniques: Recombinant, Synthesized, Diagnostic Assay, Software, Drug discovery, Flow Cytometry, Viability Assay, Histopathology