Journal: Cell death & disease

Article Title: Investigation into the role of the MITA-TRIM38 interaction in regulating pyroptosis and maintaining immune tolerance at the maternal-fetal interface.

doi: 10.1038/s41419-023-06314-w

Figure Lengend Snippet: Fig. 1 Differential expression of pyroptosis-associated markers and macrophage subpopulations in decidual tissues between patients with URSA and control patients. A Differences in the protein expression levels of GSDMD, GSDMD-N, pro-caspase-1 and mature caspase-1 in decidual tissues between patients in the URSA group and the control group. B FCM analysis shows the content of CD14-labeled macrophages, FVS-labeled live/dead cells, and the contents of M1 macrophages labeled by CD86 and M2 macrophages labeled in CD209 in viable and dead cells in the decidual tissues in one patient with URSA and one control patient. a shows that the percentages of all the macrophages with CD14 (+) in the URSA and control groups are 5.02% and 1.32%, respectively; b shows that the percentages of CD14 (+) in FVS (−) and FVS (+) in the URSA and control groups are 53.6%, 42.1%, 28.4% and 38.2%; C shows that the percentages of macrophages showing the FVS (−) survival status represented by CD86 (+) M1 macrophages in the URSA group and control group are 38.5% and 33.3%, respectively; d shows that the percentages of macrophages showing the FVS (−) survival status represented by CD209 ( + ) M2 macrophages in the URSA and control groups are 31.2% and 71.5%, respectively; e shows that the percentages of macrophages showing the FVS (+) dead status represented by CD86 (+) M1 macrophages in the URSA group and control group are 45.4% and 30.4%; f shows that the percentages of macrophages showing the FVS (+) dead status represented by CD209 (+) M2 macrophages in the URSA group and control group are 22.5% and 55.2%. C FCM analysis of differences in total macrophage content in decidual tissues between patients in the URSA group and the control group. D FCM analysis of the proportion of macrophage survival and death in the uterine decidual tissues of patients in the URSA group and the control group. E FCM analysis of the difference in the proportion of M1 and M2 macrophages in the surviving and dead macrophages of the URSA group and the control group. *P < 0.05, **P < 0.01, ns = nonsignificant.

Article Snippet: For triple staining of CD86+ CD206 and TRIM38, the sections were treated with 3% bovine serum albumin at room temperature for 30min, followed by overnight incubation at 4 °C with the primary antihuman antibody CD206 (#60143-1-Ig, Proteintech, dilution 1:400).

Techniques: Quantitative Proteomics, Control, Expressing, Labeling

Journal: Cell death & disease

Article Title: Investigation into the role of the MITA-TRIM38 interaction in regulating pyroptosis and maintaining immune tolerance at the maternal-fetal interface.

doi: 10.1038/s41419-023-06314-w

Figure Lengend Snippet: Fig. 2 Differential expression of TRIM38 and MITA in decidual tissues between patients in the URSA group and the control group as determined by immunohistochemistry and immunofluorescence staining. A Immunohistochemistry shows the differences in MITA and TRIM38 expression in the uterine decidual tissue of patients with URSA and controls (brown part). B Triple immunofluorescence staining shows the distribution and expression differences of M1 macrophages (CD86 marker, red), M2 macrophages (CD206 marker, green) and TRIM38 (pink) in the uterine decidual tissue of patients with URSA and controls. C Immunofluorescence staining shows the differences in the distribution and expression differences of M1 macrophages (CD86 marker, red), M2 macrophages (CD206 marker, green) and MITA (pink) in the uterine decidual tissue of patients with URSA and controls. D Local magnification plot corresponding to B. E Local magnification plot corresponding to C.

Article Snippet: For triple staining of CD86+ CD206 and TRIM38, the sections were treated with 3% bovine serum albumin at room temperature for 30min, followed by overnight incubation at 4 °C with the primary antihuman antibody CD206 (#60143-1-Ig, Proteintech, dilution 1:400).

Techniques: Quantitative Proteomics, Control, Immunohistochemistry, Staining, Expressing, Marker

Journal: Cell death & disease

Article Title: Investigation into the role of the MITA-TRIM38 interaction in regulating pyroptosis and maintaining immune tolerance at the maternal-fetal interface.

doi: 10.1038/s41419-023-06314-w

Figure Lengend Snippet: Fig. 7 The relationship between pyroptosis and K48. A Morphology and number of pyroptotic cells in the supernatants of wild-type M1, M1 with DMSO (M1-vehicle), M1 with VX765 (M1-VX765) and wild-type M2 macrophages under a ×40 microscope. B Differences in the protein expression levels of GSDMD-N, caspase-1 and cleaved caspase-1 in the supernatants of M1, M1 with DMSO (M1-vehicle), M1 with VX765 (M1- VX765) and M2 macrophages. C Differences in the protein expression levels of MITA and cGAS in M1 macrophages, M1 macrophages treated with DMSO (M1-vehicle), M1 macrophages treated with VX765 (M1-VX765) and M2 macrophages. D Differential expression of MITA by ubiquitination of type K48 in M1, M1 with DMSO (M1-vehicle) and M1 with VX765 (M1-VX765) macrophages. E FCM analysis shows wild-type M1, M1 with DMSO (M1-vehicle), M1 with VX765 (M1-VX765) of FVS-labeled live cells, and the content of macrophages labeled by CD86 or CD209 in viable cells in the above three kinds of cells. a–c shows that the percentages of FVS (−) in M1, M1-vehicle and M1-VX765 are 59.5%, 58.9% and 58.2%, respectively. d–f shows that the percentages of macrophages showing the FVS (−) survival status represented by CD86 (+) macrophages in M1, M1-vehicle and M1-VX765 are 49.1%, 48.2% and 42.5%, respectively. g–i shows that the percentages of macrophages showing the FVS (−) survival status represented by CD209 (+) macrophages in M1, M1-vehicle and M1-VX765 are 0.09%, 0.10% and 0.86%, respectively. F The phenotypes of CD86 and CD209 in wild-type M1, M1-vehicle, and M1-VX765 cells were analyzed by FCM. *P < 0.05, ****P < 0.0001, ns = nonsignificant.

Article Snippet: For triple staining of CD86+ CD206 and TRIM38, the sections were treated with 3% bovine serum albumin at room temperature for 30min, followed by overnight incubation at 4 °C with the primary antihuman antibody CD206 (#60143-1-Ig, Proteintech, dilution 1:400).

Techniques: Microscopy, Expressing, Quantitative Proteomics, Ubiquitin Proteomics, Labeling

Journal: Cell death & disease

Article Title: Investigation into the role of the MITA-TRIM38 interaction in regulating pyroptosis and maintaining immune tolerance at the maternal-fetal interface.

doi: 10.1038/s41419-023-06314-w

Figure Lengend Snippet: Fig. 9 Effect of lentiviral knockdown of TRIM38 or MITA on the polarization efficiency of macrophages. A FCM analysis shows the wild- type M1, shTRIM38-M1, shMITA-M1 of FVS-labeled live cells, and the content of macrophages labeled by CD86 in viable cells in the above three kinds of cells, respectively. a–c shows the percentage of FVS (−) in M1, shTRIM38-M1, and shMITA-M1 are 75.0%, 75.7% and 74.7%, respectively. d–f shows that the percentages of macrophages showing the FVS (−) survival status represented by CD86 (+) macrophages in M1, shTRIM38-M1, and shMITA-M1 are 76.5%, 86.4% and 65.3%, respectively. B The phenotypes of FVS(−) and surviving macrophages with CD86(+) in wild-type M1, shTRIM38-M1, and shMITA-M1 cells were analyzed by FCM. **P < 0.01, ns = nonsignificant. C FCM analysis shows the wild-type M2, shTRIM38-M2, and shMITA-M2 FVS-labeled live cells and the content of macrophages labeled by CD209 in viable cells in the above three kinds of cells; a–c shows that the percentages of FVS (−) in M2, shTRIM38-M2, and shMITA-M2 are 74.9%, 72.4% and 72.9%, respectively; d–f shows the percentage of macrophages showing the FVS (−) survival status represented by CD209 (+) macrophages in M2, shTRIM38-M2, and shMITA-M2 are 50.9%, 32.7% and 76.6%, respectively. D The phenotypes of FVS(−) and surviving macrophages with CD209(+) in wild-type M2, shTRIM38-M2, and shMITA-M2 cells were analyzed by FCM. ***P < 0.001, ns nonsignificant.

Article Snippet: For triple staining of CD86+ CD206 and TRIM38, the sections were treated with 3% bovine serum albumin at room temperature for 30min, followed by overnight incubation at 4 °C with the primary antihuman antibody CD206 (#60143-1-Ig, Proteintech, dilution 1:400).

Techniques: Knockdown, Labeling

Journal: Oncotarget

Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

doi: 10.18632/oncotarget.22287

Figure Lengend Snippet: ( A , B ) G-TPP leads to Ub-charging of the active site mutant Parkin C431S. HeLa cells stably expressing 3xFLAG-Parkin C431S were treated with 10 µM G-TPP for the indicated times. As positive control some cells were treated with 10 µM CCCP for 2 h. (A) Western blots were prepared with cell lysates and probed with antibodies against FLAG. Ub-charged Parkin can be observed as 8 kDa band shift that collapses again with NaOH treatment. (B) Densitometric quantification of Parkin Ub-charging normalized to 2 h CCCP treatment. Shown is the average of three independent experiments ± SEM (one-way ANOVA with Tukey’s post-hoc test, *** p < 0.0005). ( C ) HeLa cells stably expressing untagged Parkin were treated with G-TPP for the indicated times. Western blots were prepared with cell lysates and probed with antibodies against Parkin substrates (Mitofusin 1 and 2, TOM70 and Miro1). Levels of Parkin substrates initially decreased but then recovered during the time course. Levels of TRAP1 were unchanged upon G-TPP treatment.

Article Snippet: The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

Techniques: Mutagenesis, Stable Transfection, Expressing, Positive Control, Western Blot, Electrophoretic Mobility Shift Assay