Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Flow cytometry analysis of U937 cells stained with sCD22-ECD alone (red) or sCD22 precomplexed with a decoy peptide comprising the M6P-binding sites (blue) or the IGF2 site (orange) on IGF2R. (B) Time-lapse fluorescence microscopy analysis of cathepsin D trafficking to lysosomes in U937 cells treated with sCD22-Δ (black), sCD22-ECD (red), sCD22-ECD, and anti-IGF2R (blue) or saturating amounts of M6P (brown) (n = 3, ANOVA, means ± SEM). N.S., not significant. (C) Time-lapse fluorescence microscopy analysis of NPC2 trafficking to lysosomes in U937 cells treated with sCD22-Δ (black), sCD22-ECD (red), sCD22-ECD and anti-IGF2R (blue), or saturating amounts of M6P (brown) (n = 2, ANOVA, means ± SEM). (D) Western blot analysis of CTSD proteoform expression in WT and IGF2R KO U937 cells treated with sCD22-ECD or sCD22-Δ for 24 hours. Equal loading was confirmed across lanes by total protein stain (n = 3, one-way ANOVA, means ± SEM). (E) Western blot analysis of NPC2 expression in WT and IGF2R KO U937 cells treated with sCD22-ECD or sCD22-Δ for 24 hours. Equal loading was confirmed across lanes by total protein stain (n = 3, one-way ANOVA, means ± SEM). (F) Representative images of NPC2 (gray) and LAMP2 (green) expression in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (G) Proportion of NPC2+ area to LAMP2+ area in U937 cells treated with sCD22-Δ or full-length sCD22-ECD (n = 8, t test, means ± SD). (H) Representative images of IGF2R (gray) colocalization (Coloc) (yellow) with the Golgi marker GOLGA1 (red) in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (I) Proportion of IGF2R localized to the Golgi in U937 cells treated with sCD22-Δ or sCD22-ECD (n = 3 biological replicates, three to four cells quantified per replicate, t test, means ± SD). (J) Representative images of IGF2R (gray) colocalization (yellow) with the lysosomal marker LAMP1 (cyan) in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (K) Proportion of IGF2R localized to the lysosome in U937 cells treated with sCD22-Δ or sCD22-ECD (n = 3 biological replicates, two to three cells quantified per replicate, t test, means ± SD). (L) Representative images of IGF2R (gray) colocalization (yellow) with wheat germ agglutinin (WGA) cell surface staining (green) in U937 cells treated with sCD22-Δ or sCD22-ECD. Scale bar, 5 μm. (M) Proportion of IGF2R localized to the cell surface in U937 cells treated with sCD22-Δ or sCD22-ECD (n = 3 biological replicates, three to four cells quantified per replicate, t test, means ± SD). (N) Flow cytometry analysis of iMGLs treated with sCD22-Δ or sCD22-ECD, incubated with pHrodo-myelin for 24 hours, and stained with BODIPY, with corresponding histograms. (O) Quantification of phagocytosis by pHrodo-myelin mean fluorescence intensity (MFI) in iMGLs treated with sCD22-Δ or sCD22-ECD (n = 4, t test, means ± SEM). (P) Quantification of lipid droplet storage by BODIPY MFI in iMGLs treated with sCD22-Δ or sCD22-ECD (n = 4, t test, means ± SEM).

Article Snippet: Exogenous lysosomal protein recapture assay Recombinant His-tagged CTSD (R&D Systems, 1014-AS) or NPC2 (Sino Biological, 13341-H08H) was conjugated to CypHer5E NHS (GE Life Sciences) as described above.

Techniques: Flow Cytometry, Staining, Binding Assay, Fluorescence, Microscopy, Western Blot, Expressing, Marker, Incubation

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of mAb generation and screening pipeline. (B) Screening results of 38 mAb clones for binding to CD22 (first column) and blocking of sCD22 to IGF2R on cell surface (second and third columns are two independent experiments). Three clones with adequate binding and potent blocking are highlighted (M22, M28, and M42). (C) Association-dissociation curves of antibody candidates binding to CD22 determined by biolayer interferometry. (D) Dose-response curves of CD22-IGF2R blockade by antibody candidates determined by flow cytometry. IC50, median inhibitory concentration. (E) Time-lapse fluorescence microscopy analysis of NPC2 trafficking to lysosomes in U937 cells treated with sCD22-Δ (gray), sCD22-ECD and an isotype control antibody (purple), or sCD22-ECD and clone M42 (green) (n = 2, ANOVA, means ± SEM). (F) Schematic of pipeline to generate isogenic WT and I1061T mutant iMGLs from iPSCs edited by CRISPR-Cas9–directed homologous recombination. After introduction of donor single-stranded DNA (ssDNA) by electroporation, a homozygous T3182C nucleotide substitution was confirmed by Sanger sequencing. NPC1 reduction was confirmed by Western blot. Mutant and isogenic control iPSCs were subsequently directed toward a hematopoietic lineage and differentiated into microglia-like cells. (G) Western blot quantification of NPC1 expression normalized to a loading control (β-actin) in WT and I1061T mutant iPSCs (n = 3, t test, means ± SEM). (H) Representative images of WT and I1061T mutant iMGLs stained for Filipin III (red, unesterified cholesterol) and IBA1 (green, microglia marker). Scale bar, 20 μm. (I) Quantification of Filipin-positive area normalized to total IBA1-positive area in WT (gray) and I1061T mutant (blue) iMGLs (n = 5 biological replicates, t test, means ± SEM). (J) Schematic of human in vitro model of microglia in NPC. Three components (iPSC-derived microglia, I1061T patient mutation, and NPC patient CSF) were combined to test the proof-of-principal in vitro efficacy of anti-CD22 in NPC. (K) Representative images of I1061T mutant iMGLs treated with NPC CSF and an isotype control antibody stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (L) Representative images of I1061T mutant iMGLs treated with NPC CSF and anti-CD22 stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (M) Quantification of Filipin-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (N) Quantification of LAMP2-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (O) Heatmap of normalized counts (z score) for differentially expressed genes in WT and I1061T mutant iMGLs treated with NPC CSF and isotype or anti-CD22. (P) Gene Ontology (GO) biological process enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. IRE1, inositol-requiring enzyme 1; IFN-γ, interferon-γ; UPR, unfolded protein response. (Q) GO cellular component enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. MHC-II, major histocompatibility complex class II; ER, endoplasmic reticulum.

Article Snippet: Exogenous lysosomal protein recapture assay Recombinant His-tagged CTSD (R&D Systems, 1014-AS) or NPC2 (Sino Biological, 13341-H08H) was conjugated to CypHer5E NHS (GE Life Sciences) as described above.

Techniques: Clone Assay, Binding Assay, Blocking Assay, Flow Cytometry, Concentration Assay, Fluorescence, Microscopy, Mutagenesis, CRISPR, Homologous Recombination, Electroporation, Sequencing, Western Blot, Expressing, Staining, Marker, In Vitro, Derivative Assay

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: bioRxiv

Article Title: Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling

doi: 10.1101/2024.03.31.587500

Figure Lengend Snippet: ( A ) Depiction of canonical via Smoothened (SMO) and non-canonical activation of Gli2. ( B ) Spearman correlation of Gli2 expression with WNT5A, FOXL1, and FOXC2 in human melanoma based on TCGA. ( C ) Expression of FoxL1 and FoxC2 in tumor tissues from an autochthonous melanoma model following anti-PD-1 antibody escape based on RNAseq (n = 3 tumors/group). ( Left ) Expression of FOXL1 and FOXC2 in human melanoma tissues based on RNAseq ( Right ) ( D ) Western blot of the active form of Gli2 after anti-PD-1 escape in the murine BRAF V600E PTEN −/− melanoma model. Representative of 3 independent experiments. ( E ) Transcriptional Nanostring analysis of melanoma tissues derived from stage IV melanoma patients undergoing anti-PD-1 therapy. R, responder. LR, late relapse. NR, nonresponder. ab, antibody. Data presented as mean ± SEM. Two group comparisons analyzed based on unpaired student’s t test. * p <0.05, *** p <0.001, **** p <0.0001.

Article Snippet: The following antibodies were used: IDO1 (mIDO-48, Santa Cruz Biotechnology, sc-53978), Gli2 (Abcam, ab7195, ab277800), Zeb1 (Cell Signaling, D80D3), Fn1 (Thermo Fisher, MA5-11981), Wnt5a (Santa Cruz, sc-365370), Cox2 (Cell Signaling, D5H5 clone, 12282), FoxL1 (invitrogen, PA5-40518), FoxC2 (Proteintech, 23066-1-AP), CXCL5 (Novus/R&D, AF433), Vimentin (Santa Cruz, sc-373717), Actin (Cell Signaling, 3700S).

Techniques: Activation Assay, Expressing, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling

doi: 10.1101/2024.03.31.587500

Figure Lengend Snippet: ( A ) Western blot analysis of Wnt5a in a BRAF V600E PTEN −/− Gli2 CA melanoma cell line. ( B ) Western blot analysis of Wnt5a in a wild-type BRAF V600E PTEN −/− melanoma cell line treated with the direct Gli2 inhibitor, Gant61. ( C ) Gene Ontogeny (GO) Pathway Analysis of the BRAF V600E PTEN −/− Gli2 CA melanoma cell line compared to a control BRAF V600E PTEN −/− cell line (NTC). Number of significantly upregulated genes adjacent to GO term. ( D ) Western blot analysis of FoxC2 and FoxL1 transcription factors in the BRAF V600E PTEN −/− Gli2 CA melanoma cell line. ( E ) Tumor growth measurements of BRAF V600E PTEN −/− NTC and Gli2 CA melanoma cell lines in C57Bl/6 mice. Flow cytometric analysis of tumor infiltrating ( F ) CD3 + CD8 + T cells, ( G ) PMN-MDSCs, and ( H ) ratio of cDC2s to cDC1s. (n = 6 tumors/group) ( I ) Whole tumor transcriptional analysis of BRAF V600E PTEN −/− NTC and BRAF V600E PTEN −/− Gli2 CA melanomas. NTC, non-target control. CA, constitutively active. Data presented as mean ± SEM. All data representative of 2-3 independent experiments. Data analyzed by E,F,G,H: unpaired student’s t test. * p <0.05 *** p <0.001.

Article Snippet: The following antibodies were used: IDO1 (mIDO-48, Santa Cruz Biotechnology, sc-53978), Gli2 (Abcam, ab7195, ab277800), Zeb1 (Cell Signaling, D80D3), Fn1 (Thermo Fisher, MA5-11981), Wnt5a (Santa Cruz, sc-365370), Cox2 (Cell Signaling, D5H5 clone, 12282), FoxL1 (invitrogen, PA5-40518), FoxC2 (Proteintech, 23066-1-AP), CXCL5 (Novus/R&D, AF433), Vimentin (Santa Cruz, sc-373717), Actin (Cell Signaling, 3700S).

Techniques: Western Blot, Control

Journal: Journal of cell science

Article Title: Rab2a and Rab27a cooperatively regulate the transition from granule maturation to exocytosis through the dual effector Noc2.

doi: 10.1242/jcs.195479

Figure Lengend Snippet: Figure 2. Rab2a interacts with the Noc2-Rab27a binary complex

Article Snippet: The following commercially purchased antibodies were also used: rabbit polyclonal antibodies toward FLAG (F7425, Sigma-Aldrich), hemagglutinin (HA; 561, MBL), green fluorescent protein (GFP; 598, MBL), Rab27a/b (18975, IBL), Noc2 (15297-1-AP, Proteintech), Rab2a (15420-1-AP, Proteintech), ICA69 (ab81500, Abcam), and PICK1 (ab3420, Abcam); and mouse monoclonal antibodies toward Rab3 (610379, BD Biosciences), EEA1 (610457, BD Biosciences), TGN38 (610849, BD Biosciences), PDI (MA3-018, Affinity BioReagents), α-tubulin (T5168, Sigma-Aldrich), and proinsulin (clone 3A1; ab8301, Abcam).

Techniques:

Journal: Journal of cell science

Article Title: Rab2a and Rab27a cooperatively regulate the transition from granule maturation to exocytosis through the dual effector Noc2.

doi: 10.1242/jcs.195479

Figure Lengend Snippet: Figure 3. The N-terminal region of Noc2 is required for binding to Rab2a

Article Snippet: The following commercially purchased antibodies were also used: rabbit polyclonal antibodies toward FLAG (F7425, Sigma-Aldrich), hemagglutinin (HA; 561, MBL), green fluorescent protein (GFP; 598, MBL), Rab27a/b (18975, IBL), Noc2 (15297-1-AP, Proteintech), Rab2a (15420-1-AP, Proteintech), ICA69 (ab81500, Abcam), and PICK1 (ab3420, Abcam); and mouse monoclonal antibodies toward Rab3 (610379, BD Biosciences), EEA1 (610457, BD Biosciences), TGN38 (610849, BD Biosciences), PDI (MA3-018, Affinity BioReagents), α-tubulin (T5168, Sigma-Aldrich), and proinsulin (clone 3A1; ab8301, Abcam).

Techniques: Binding Assay

Journal: Journal of cell science

Article Title: Rab2a and Rab27a cooperatively regulate the transition from granule maturation to exocytosis through the dual effector Noc2.

doi: 10.1242/jcs.195479

Figure Lengend Snippet: Figure 6. Effects of overexpression of Noc2 and its mutants on insulin secretion

Article Snippet: The following commercially purchased antibodies were also used: rabbit polyclonal antibodies toward FLAG (F7425, Sigma-Aldrich), hemagglutinin (HA; 561, MBL), green fluorescent protein (GFP; 598, MBL), Rab27a/b (18975, IBL), Noc2 (15297-1-AP, Proteintech), Rab2a (15420-1-AP, Proteintech), ICA69 (ab81500, Abcam), and PICK1 (ab3420, Abcam); and mouse monoclonal antibodies toward Rab3 (610379, BD Biosciences), EEA1 (610457, BD Biosciences), TGN38 (610849, BD Biosciences), PDI (MA3-018, Affinity BioReagents), α-tubulin (T5168, Sigma-Aldrich), and proinsulin (clone 3A1; ab8301, Abcam).

Techniques: Over Expression