Journal: Research

Article Title: USP18 Antagonizes Pyroptosis by Facilitating Selective Autophagic Degradation of Gasdermin D

doi: 10.34133/research.0380

Figure Lengend Snippet: E3 ubiquitin ligase MIB2 enhances GSDMD degradation via mediating the ubiquitination of GSDMD at K168. (A and B) HEK293T cells were transfected with HA-GSDMD and increasing amounts of Flag-MIB2. Cell lysates were collected for immunoblot analysis (A). Quantification of the relative protein level of GSDMD was shown in (B). (C to F) THP-1-derived macrophages were transfected with control siRNA (si Ctrl ) or MIB2 siRNA (si MIB2 ) for 48 h, pre-treated with LPS (200 ng/ml) for 3 h, then treated with DMSO (vehicle), MG132 (10 μM) or carfilzomib (100 nM), 3-methyladenine (3-MA, 10 mM), bafilomycin A1 (Baf A1, 0.2 μM), or chloroquine (CQ, 50 μM) for 6 h. Cell lysates were collected for immunoblot analysis (C and E). Quantitative analysis of the relative protein level of GSDMD was shown in (D) and (F), respectively. (G and H) THP-1-derived macrophages were transfected with si Ctrl or si MIB2 (#1 and #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment. Cell lysates were collected for immunoprecipitation and immunoblot analysis (G). Quantitative analysis of the relative immunoprecipitated level of ubiquitin (Ub) was shown in (H). (I and J) HEK293T cells were transfected with HA-Ub and wild type (WT) Flag-GSDMD or Flag-GSDMD K168R, together with Myc-empty vector (EV) or Myc-MIB2 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for immunoprecipitation and immunoblot analysis (I). Quantitative analysis of the relative immunoprecipitated level of HA-Ub was shown in (J). (K and L) Immunoblot analysis of HEK293T cells transfected with Flag-GSDMD WT or Flag-GSDMD K168R mutant, together with HA-EV or HA-MIB2 (K). Quantitative analysis of the relative protein level of GSDMD was shown in (L). (M) In vitro binding assay of 6×His-MIB2 and Flag-GSDMD proteins purified from HEK293T cells, respectively. (N) In vitro ubiquitination assay was performed in the presence of Ub, E1, E2 (UbcH5a), Flag-GSDMD, and 6×His-MIB2. (O to Q) THP-1-derived macrophages were transfected with si Ctrl or si MIB2 (#1 and #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment. Cell lysates were collected for immunoblot analysis (O). Cell death (P) and production of IL-1β (Q) were assessed by LDH (lactate dehydrogenase) release assay and ELISA analysis in the supernatants, respectively. In (A), (C), (E), (G), (I), (K) and (M) to (O), data are representative of 3 independent experiments with similar results. In (B), (D), (F), (H), (J), and (L), quantification of the indicated protein levels was determined by Image Lab software, data are presented as mean values ± SD, and P values were determined by unpaired 2-tailed Student’s t test of n = 3 independent biological experiments. In (P) and (Q), data are presented as mean values ± SEM and P values were determined by unpaired 2-tailed Student’s t test of n = 3 independent biological experiments.

Article Snippet: Briefly, purified Flag-GSDMD was incubated with ubiquitin (Ub), E1 activating enzyme, Mg-ATP, and E2 enzyme (UbcH5a, MCE, Cat# HY-P71399) with or without purified 6×His-MIB2 protein in ubiquitinoylation buffer in a total 50-μl reaction volume at 37 °C for 3 h. The assays were quenched by the addition of 50 μl of 2× non-reducing gel loading buffer, and 20 μl of each quenched assay was resolved by SDS-PAGE.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Derivative Assay, Control, Immunoprecipitation, Plasmid Preparation, Mutagenesis, In Vitro, Binding Assay, Purification, Release Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: UBE2D family proteins redundantly promote the ubiquitination of GSPT1 (A) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that the amino acid sequence identity among all 4 family proteins is close to 90%. (B) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4CRBN complex in the presence of UBE2G1, UBE2D1, UBE2D2, or UBE2D3, alone or in combination. Recombinant protein products as indicated were incubated with or without 80 μM CC-885 in the ubiquitination assay buffer at 30 °C for 2 hours, and then analyzed by immunoblotting. SE, short exposure; LE, long exposure

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: Sequencing, In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot

Journal: Nature Communications

Article Title: Ventral hippocampus neurons encode meal-related memory

doi: 10.1038/s41467-025-59687-1

Figure Lengend Snippet: a Left: Diagram of a coronal section of the nucleus accumbens (ACB), adapted from the Brain Maps 4.0 structure of the rat brain . Middle: Representative photomicrographs of axonal green fluorescent protein (GFP) + expression in the ACB from ventral CA1 neurons active in the Fasted (Fasted GFP )( n = 5) or Fed (Fed GFP )( n = 4) state, relative to the anterior commissure (ac); scale bar 100 μm. Right: Average number of GFP + pixels in the ACB. b Left: Diagram of a coronal section of the lateral septum (LS), adapted from the Brain Maps 4.0 structure of the rat brain . Middle: Representative photomicrographs of axonal GFP + expression in the LS from ventral CA1 neurons active in the Fasted (Fasted GFP )( n = 5) or Fed (Fed GFP )( n = 4) state, relative to the lateral ventricle (lv); scale bar 100 μm. Right: Average number of GFP + pixels in the LS. c Left: Diagram of a coronal section of the lateral hypothalamic area (LHA), adapted from the Brain Maps 4.0 structure of the rat brain . Middle: Representative photomicrographs of axonal GFP + expression in the LHA from ventral CA1 neurons active in the Fasted (Fasted GFP )( n = 5) or Fed (Fed GFP )( n = 4) state, relative to the fornix (fx) and 3rd ventricle (3 v); scale bar 100 μm. Right: Average number of GFP + pixels in the LHA ( p value = 0.0159). d Left: Representative photomicrograph of retrograde GFP viral expression in the LS, relative to the lv; scale bar 200 μm. Right: Representative photomicrograph of retrograde mCherry viral expression in the LHA, relative to the fx; scale bar 200 μm. e Average number of LS (GFP)( n = 4) and LHA (mCherry)( n = 4)-projecting neurons labeled in the CA1v. f Average percentage of CA1v neurons sending axonal projections to both the LS (GFP)( n = 4) and LHA (mCherry)( n = 4). g Average percentage of LS (GFP)( n = 4) and LHA (mCherry)( n = 4)-projecting CA1v neurons expressing cFos in the Fed state ( p value < 0.0001). h Representative photomicrograph of fluorescent in situ hybridization for Fos (cyan) in LS (GFP) and LHA (mCherry)-projecting neurons of the CA1v, relative to the alveus (alv); scale bar 100 μm. Data are presented as mean ± SEM. For a – c and d – g , two-tailed unpaired t-test; * p < 0.05, **** p < 0.0001. No symbol indicates lack of significance.

Article Snippet: The same rats also received a unilateral injection (200nL) of a retrograde AAV expressing mCherry (AAVrg-hSyn-mCherry; Addgene viral prep # 11472-AAVrg gifted by Karl Deisseroth) into the LHA at the following coordinates: −2.9 mm AP, + or −1.1 mm ML, and −8.6 mm DV (all defined at bregma).

Techniques: Expressing, Labeling, In Situ Hybridization, Two Tailed Test

Journal: Nature Communications

Article Title: Ventral hippocampus neurons encode meal-related memory

doi: 10.1038/s41467-025-59687-1

Figure Lengend Snippet: a Diagram of viral approach for expression of GCaMP7s in ventral CA1 neurons projecting to the lateral hypothalamic area and implantation of the optic fiber, adapted from the Brain Maps 4.0 structure of the rat brain . b Representative photomicrograph of GCaMP7s expression for placement validation of viral injections, relative to the alveus (alv); scale bar 100 μm (representative pattern was observed in all animals used in the analyses, n = 3). c Representative trace of a single animal of the increase in calcium-dependent activity during the interbout intervals (purple) in ventral hippocampus neurons projecting to the lateral hypothalamic area. d Average change in z-score for fluorescence over the course of an eating bout versus during an interbout interval ( n = 3 animals)( p value = 0.0165). e Diagram of viral approach for expression of hM4Di receptors in ventral CA1 neurons projecting to the lateral hypothalamic area and administration of vehicle (VEH) or clozapine-N-oxyde (CNO) through a lateral ventricle (LV) cannula, adapted from the Brain Maps 4.0 structure of the rat brain . f Representative photomicrograph of mCherry expression for placement validation of viral injections, relative to the alveus (alv); scale bar 100 μm (representative pattern was observed in all animals used in the analyses, n = 10). g Average number of errors and h latency to find the food during training for the foraging-related spatial memory task in animals assigned to VEH ( n = 6) or CNO ( n = 4) groups. i Performance index during the probe for the foraging-related spatial memory task, 1 h following LV administration of VEH ( n = 6) or CNO ( n = 4)(Group comparison p value = 0.0024; p value for 1-sample t-test = 0.0014 for VEH group). Data are presented as mean ± SEM. For d , two-tailed paired t-test. For g – h , two-way ANOVA. For i , two-tailed unpaired t-test ( n = 4–6/group); * p < 0.05, ** p < 0.01. For i , one-sample t-test, different from chance set at 0.1667; ## p < 0.01. No symbol indicates lack of significance.

Article Snippet: The same rats also received a unilateral injection (200nL) of a retrograde AAV expressing mCherry (AAVrg-hSyn-mCherry; Addgene viral prep # 11472-AAVrg gifted by Karl Deisseroth) into the LHA at the following coordinates: −2.9 mm AP, + or −1.1 mm ML, and −8.6 mm DV (all defined at bregma).

Techniques: Expressing, Biomarker Discovery, Activity Assay, Fluorescence, Comparison, Two Tailed Test