Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a C4a and C4b restore the astrocytic engulfment of microglial debris in vitro in serum-free culture medium. b Quantifications of the phagocytic influence by complement supplementation and preopsonization in serum-free culture medium. N = 11 independent biological replicates of each group. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). c Scheme of in vivo microglial depletion and time points for analysis. d Reanalysis of RNA-seq data from whole-brain homogenate (GSE108269 ) showing that Gfap and C4b are upregulated and C1qa is downregulated during microglial ablation, whereas C2 , C3 and C4a remain at low levels and are unaffected. N = 5 mice at D0 and N = 4 mice at D2 to D21. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). e qPCR further confirmed the upregulation of C4b in sorted astrocytes upon microglial depletion. N = 5 in each group. Two-tailed independent t test. f Scheme of the in vivo examination of astrocytic engulfment using AAV PHP.eB-based astrocyte labeling and microglial depletion. g Confocal orthogonal colocalization and 3D reconstruction show that C4b −/− impairs the astrocytic engulfment of microglial debris under physiological condition (D21) and upon CSF1R inhibition (D23). h Quantification of microglial debris engulfment by astrocytes. N = 7 (D21) and 8 (D23) WT mice, and N = 3 (D21) and 5 (D23) C4b −/− mice. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). PLX5622 PLX5622-formulated AIN-76A diet, CD control AIN-76A diet, IV intravenous, MFI mean fluorescence intensity, Ctx cortex, Hipp hippocampus, OB olfactory bulb. Data are presented as mean ± SD. The source data are provided as a Source Data file.

Article Snippet: Recombinant mouse complement component C2 protein CF (C2) was acquired from R&D Systems (Cat#: 6725-SE-010).

Techniques: In Vitro, In Vivo, RNA Sequencing, Two Tailed Test, Labeling, Inhibition, Control, Fluorescence

Journal: Nature Communications

Article Title: DSS1 inhibits autophagy to activate epithelial-mesenchymal transition in a pro-metastatic niche of renal cell carcinoma

doi: 10.1038/s41467-025-62135-9

Figure Lengend Snippet: a Identification of TWIST1 as a potential downstream effector of DSS1-autophagy axis. Gene signatures (Molecular Signature DataBase v2022.1) were analyzed by single-sample gene set enrichment analysis (ssGSEA) in TCGA-KIRC ( n = 522 distinct tumors). b Fold changes (DESeq2) of transcription factor (TF) mRNA expression in metastatic (Met.) vs. primary ccRCC and primary ccRCC vs. normal kidney (TCGA-KIRC). c Immunoblotting of EMT-TFs (ZEB1, ZEB2, SNAI1, SLUG/SNAI2, FOXC2, TCF3) in sh DSS1 vs. sh NC cells (two-tailed Welch’s t-test, Benjamini-Hochberg [BH] adjustment). β-actin: endogenous control. d Immunoblotting showing TWIST1 protein levels in cells treated with si NC /si DSS1 , autophagy inhibitor (CQ, 10 μM; Control: 0.02% DMSO; 24 h before harvest), or both (two-tailed Welch’s t-test). e Immunoblotting showing TWIST1 levels in cells treated with si NC , TWIST1 knockdown, pcDNA3.1 or TWIST1 plasmids (two-tailed Welch’s t-test). f Rescue experiments showing protein levels of EMT markers in ccRCC cells transfected with different combinations of control, DSS1 , and TWIST1 siRNAs, and DSS1 and TWIST1 plasmids (two-tailed Welch’s t-test). g Transwell assays following the same treatments as in panel ( f ) (scale bar: 100 μm, two-tailed Welch’s t-test). h Immunohistochemistry using lung metastatic lesions of xenograft mouse models generated by tail vein injection of Caki-1 cells treated with Lv-sh DSS1 or control Lv-sh NC ( n = 5 independent samples per group; scale bar: 100 μm; two-tailed Welch’s t-test). IOD, Integrated Optical Density. i Caki-1 cells were analyzed by immunoprecipitation with antibody to the LC3 or TWIST1 epitope, followed by SDS-PAGE and immunoblotting (IgG: negative control, input: 5% lysate). j Immunofluorescence microscopy showing the co-localization of Bcl-2 (magenta), p62 (orange), and TWIST1 (turquoise) in ACHN cells treated with DSS1 knockdown or control (scale bar: 5 μm). DAPI (blue): nucleus. c –f The samples derived from the same experiment were run on parallel gels, with each gel probed for a different antibody. c – g , i , j n = 3 independent experiments. c – h Error bars: mean ± SD. Statistics are provided in the source data. Source data are provided as a Source Data file.

Article Snippet: After blocking in 5% bovine serum albumin or 5% nonfat milk at RT for 2 h, the membranes were incubated with primary antibodies against DSS1 (Proteintech, 13639-1-AP, 1:1000), CDH1/E-cadherin (Proteintech, 20874-1-AP, 1:1000), CDH2/N-cadherin (Proteintech, 22018-1-AP, 1:1000), VIM/Vimentin (Proteintech, 10366-1-AP, 1:1000), LC3 (Proteintech, 14600-1-AP, 1:1000), SQSTM1/p62 (Proteintech, 18420-1-AP, 1:1000), TWIST1 (Proteintech, 25465-1-AP, 1:1000), TRIM25 (Proteintech, 12573-1-AP, 1:1000), ubiquitin (Abcam, ab134953, 1:1000), Myc-tag (Proteintech, 16286-1-AP, 1:1000), HA-tag (Proteintech, 51064-2-AP, 1:1000), mTOR (Abways, CY5306, 1:1000), p-mTOR (Abways, CY5996, 1:1000), SPP1 (Abcam, ab214050, 1:1000), Bcl-2 (Abways, CY5032, 1:1000), Beclin 1 (HUABIO, HA721216, 1:1000), TGF-β (Immunoway, YM8257, 1:1000), ZEB1 (Cell Signaling Technology, 70512, 1:1000), ZEB2 (Cell Signaling Technology, 97885, 1:1000), SNAI1 (Proteintech, 13099-1-AP, 1:1000), SNAI2/SLUG (Cell Signaling Technology, 9585, 1:1000), TCF3 (Proteintech, 21242-1-AP, 1:1000), FOXC2 (Proteintech, 23066-1-AP, 1:1000), GST-tag (Proteintech, 66001-2-Ig, 1:1000) and ACTB/β-actin (Proteintech, 20536-1-AP, 1:1000) at 4 °C overnight.

Techniques: Expressing, Western Blot, Two Tailed Test, Control, Knockdown, Transfection, Immunohistochemistry, Generated, Injection, Immunoprecipitation, SDS Page, Negative Control, Immunofluorescence, Microscopy, Derivative Assay