Journal: American Journal of Cancer Research

Article Title: Tumor-associated macrophages promoting PD-L1 expression in infiltrating B cells through the CXCL12/ CXCR4 axis in human hepatocellular carcinoma

doi: 10.62347/ziax8828

Figure Lengend Snippet: Figure 2. High PD-L1 expression Bregs were mainly located in adjacent HCC tissues and associated with exhausted CTLs in human HCC. A. Representative figures (×200 magnification) of multiple-color staining for CD38, PD-L1, and IgA expression in human HCC and adjacent tissue. B. Plots showing the number (left panel) and percentage (right panel) of IgA+PD-L1+ Breg cells in human HCC and adjacent tissue. C. A quantitative comparison of B cells and IgA+ PD-L1+ B cells in HCC and adjacent tissue using multiple-color staining. Data are presented as means ± SEM and were analyzed with Student’s t-test (**, P<0.01; ns, no significance, P>0.05). D. Comparison and gating strategy

Article Snippet: The sections were stained with antibodies against human Tim3 (1:200; Signalway Antibody LLC, #28301), CD8 (1:200, Cell Signaling Technology, C8/144B), CD163 (1:200, Cell Signaling Technology, D6U1J), CD38 (1:200, Proteintech, 25284-1-AP), IgA (1:200, Proteintech, 11449-1-AP), CXCL12 (1:200, Proteintech, 17402-1-AP), CXCR4 (1:50, Abcam, 60042-1-Ig), PD-L1 (1:40, Abcam, ab58810), HRP-conjugated anti-mouse or anti-rabbit IgG (PANOVUE), and PPD 520/ 540/570/620/650/690/DAPI (PANOVUE).

Techniques: Expressing, Staining, Comparison

Journal: American Journal of Cancer Research

Article Title: Tumor-associated macrophages promoting PD-L1 expression in infiltrating B cells through the CXCL12/ CXCR4 axis in human hepatocellular carcinoma

doi: 10.62347/ziax8828

Figure Lengend Snippet: Figure 7. CXCL12-secreting TAMs regulate PD-L1+ Bregs through the CXCL12/CXCR4 axis. A. Representative figures for multiple-color staining of CD38, IgA, PD-L1, CXCR4, CD163, and CXCL12 expression in human HCC and adjacent samples. B. Dot plots showing the proportions of four types of cells. Different colors represent different cells. C. Liner correlation studies were performed for CXCR4 expression vs PD-L1 expression in PD-L+ CXCR4+ B cells and B cells in HCC and adjacent tissues. D. Linear correlation studies were performed between a count of TAMs and CXCL12+ TAMs in clinical samples with a count of B cells, PD-L1+ Breg cells, and PD-L1+ CXCR4+ B cells.

Article Snippet: The sections were stained with antibodies against human Tim3 (1:200; Signalway Antibody LLC, #28301), CD8 (1:200, Cell Signaling Technology, C8/144B), CD163 (1:200, Cell Signaling Technology, D6U1J), CD38 (1:200, Proteintech, 25284-1-AP), IgA (1:200, Proteintech, 11449-1-AP), CXCL12 (1:200, Proteintech, 17402-1-AP), CXCR4 (1:50, Abcam, 60042-1-Ig), PD-L1 (1:40, Abcam, ab58810), HRP-conjugated anti-mouse or anti-rabbit IgG (PANOVUE), and PPD 520/ 540/570/620/650/690/DAPI (PANOVUE).

Techniques: Staining, Expressing

Journal: Cell research

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

doi: 10.1038/s41422-019-0141-z

Figure Lengend Snippet: Fig. 1 The PARylation levels influence the assembly-disassembly dynamics of SGs containing ALS-related RNPs. a–c PARP inhibition suppresses SG assembly. a Representative confocal images of HeLa cells treated with PBS (no stress) or 100 μM arsenite (stress) for the indicated time in the absence (DMSO) or presence of the PARP inhibitor Olaparib (20 μM). All cells are transfected with TDP-43-HA (anti-HA) and stained for SGs by the anti-TIAR (arrowheads); the merged images with the DAPI staining of DNA (blue) are shown. b The percentage of cells with SGs and TDP-43 + SGs from a is quantified. c Western blot analysis confirms the decrease in the overall PARylation levels by Olaparib, with GAPDH as a loading control. d–f Hydrolysis of PAR by PARG is required for SG disassembly. d Cells transfected with the siRNA (si-PARG) or the scrambled siRNA (si-Ctrl) are stressed with arsenite (100 μM) for 30 min and then washed for the indicated time. All cells are transfected with the hnRNP A1-Flag (anti-Flag) and stained for SGs with the anti-TIAR (arrowheads); the merged images with the DAPI staining for DNA (blue) are shown. e Percentage of the cells with SGs or hnRNP A1 + SGs from (d); f Western blot analysis confirms PARG KD and the increase in the overall PARylation levels in the cells by si-PARG. Data are shown as the mean ± SEM; n = over 100 cells for each condition, pooled results of three independent repeats; *p < 0.05, **p < 0.01; ns, not significant; ud, undetected, Student’s t-test. Scale bars 5 μm

Article Snippet: In vitro liquid-liquid phase separation (LLPS) assay For the LLPS assay, purified hnRNP A1 or TDP-43 protein were mixed with PAR polymers (Trevigen, 4336–100–01) and NaCl at the indicated concentrations in a LLPS buffer (50 mM Tris-HCl, pH 7.5, 10% (w/v) PEG 3550 (Sigma) and 2mM DTT) and incubated for 3 min at room temperature.

Techniques: Inhibition, Transfection, Staining, Western Blot, Control

Journal: Cell research

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

doi: 10.1038/s41422-019-0141-z

Figure Lengend Snippet: Fig. 2 PARylation and PAR binding of TDP-43 and hnRNP A1 in vitro and in cells. a, b HA-tagged TDP-43 (a) or Flag-tagged hnRNP A1 (b) is expressed and immunoprecipitated from HeLa cells that are treated with or without H2O2, and then examined by Western blotting with indicated antibodies. hnRNP A1 shows both steady state and induced levels of PARylation, whereas no PARylation of TDP-43 was detected even when the blot is overexposed to facilitate the detection. c Cells expressing the Flag-tagged hnRNP A1 are treated with H2O2 and immunoprecipitated with the anti-Flag as in (b); PARG is added to the immunoprecipitates and then examined by Western blot with anti-Flag or anti-PAR. The bands of hnRNP A1 and the associated proteins in the anti-PAR blot are drastically decreased, confirming that they are indeed due to PARylation. d Purified, recombinant His-tagged TDP-431–274, TDP-43274–414 and full-length hnRNP A1 proteins (4 μg) were subjected to in vitro PARylation with the recombinant PARP1 and ssDNA (to activate PARP1 in vitro) as indicated. After reaction, proteins were separated on the SDS-PAGE gel. The Coomassie Brilliant Blue staining confirms equal protein loading (upper panel), followed by immunoblotting with the anti-PAR (lower panel). Activated PARP1 shows self-PARylation smears at above 115 kDa, which induces PARylation of TDP-431–274 and hnRNP A1 but not TDP-43274–414. e A diagram showing the heterogeneity of PARylation, which is reversibly catalyzed by PARPs and PARG. ADPr, an ADP-ribose unit. f Functional domains of the human hnRNP A1 protein. RRM RNA recognition motif, RGG RGG box RNA binding domain, M9 a nuclear targeting sequence termed M9, GRD glycine-rich domain, PBM PAR-binding motif. The consensus sequence and the residues mutated in the PBM and putative PARylation sites are indicated. H hydrophobic amino acid residues, B basic amino acid residues. g Examination of PARylation and PAR-binding of the WT, K298A and PBMmut hnRNP A1. hnRNP A1-Flag was immunoprecipitated from HeLa cells with the anti- Flag and examined by Western blotting with the anti-PAR. The blot is overexposed to facilitate the detection of less abundant PARylated proteins. * the rabbit anti-HA IgG heavy chain, # PARylated proteins co-immunoprecipitated with hnRNPA1. h–i Representative images (h) and quantification (i) of the dot-blot assay showing significantly reduced binding affinity of the PBMmut to the PAR polymer in vitro. The MBP was used as a negative control. The PAR intensity was normalized to that of the Ponceau S staining (loading control) and shown as the percentage to the WT hnRNP A1 in (i). Mean ± SEM, n = 3, ***p < 0.001; Student’s t-test

Article Snippet: In vitro liquid-liquid phase separation (LLPS) assay For the LLPS assay, purified hnRNP A1 or TDP-43 protein were mixed with PAR polymers (Trevigen, 4336–100–01) and NaCl at the indicated concentrations in a LLPS buffer (50 mM Tris-HCl, pH 7.5, 10% (w/v) PEG 3550 (Sigma) and 2mM DTT) and incubated for 3 min at room temperature.

Techniques: Binding Assay, In Vitro, Immunoprecipitation, Western Blot, Expressing, Recombinant, SDS Page, Staining, Functional Assay, RNA Binding Assay, Sequencing, Dot Blot, Polymer, Negative Control, Control

Journal: Cell research

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

doi: 10.1038/s41422-019-0141-z

Figure Lengend Snippet: Fig. 3 The PARylation and PAR-binding mutants of hnRNP A1 K298A and PBMmut exhibit deficits in translocation and targeting to SGs. a Representative images of HeLa cells transfected with the WT, K298A or PBMmut of the Flag-tagged hnRNP A1 and treated with PBS (no stress) or arsenite (stress) for 30 min. Arrows, hnRNP A1 + /TIAR- cytoplasmic foci; arrowheads, hnRNP A1 + /TIAR + SGs; *, hnRNP A1-/TIAR + SGs. b, c The percentage of cells showing SGs (TIAR + ) (b) or SGs co-localized with hnRNP A1 (hnRNP A1 + /TIAR + ) (c) is quantified. d, e The occurrence of abnormal hnRNP A1 cytoplasmic foci is assessed as the average percentage of cells showing hnRNP A1 + /TIAR- foci (d). The average count of hnRNP A1 + /TIAR- foci per cell was evaluated in such cells (e). Mean ± SEM except for (e), for which the box-and-whisker plots are displayed; n = over 300 cells for each group, pooled results of three independent repeats; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant; Student’s t-test for comparison between stress and no stress within the same genotype, and two-way ANOVA for comparison of the stress-induced changes across different genotypes. Scale bar 5 μm

Article Snippet: In vitro liquid-liquid phase separation (LLPS) assay For the LLPS assay, purified hnRNP A1 or TDP-43 protein were mixed with PAR polymers (Trevigen, 4336–100–01) and NaCl at the indicated concentrations in a LLPS buffer (50 mM Tris-HCl, pH 7.5, 10% (w/v) PEG 3550 (Sigma) and 2mM DTT) and incubated for 3 min at room temperature.

Techniques: Binding Assay, Translocation Assay, Transfection, Whisker Assay, Comparison

Journal: Cell research

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

doi: 10.1038/s41422-019-0141-z

Figure Lengend Snippet: Fig. 4 PAR promotes LLPS of hnRNP A1 in vitro. a hnRNP A1 forms dynamic LDs by LLPS in vitro. The concentrations of hnRNP A1 and NaCl are shown. The higher hnRNP A1 and lower NaCl concentrations, the more and larger LDs are formed. b Addition of PAR promotes LLPS of hnRNP A1 in a dose-dependent manner. c The same concentration of heparin (7 μM) does not induce LLPS of hnRNP A1 at the same condition. d PAR (7 μM) alone does not phase separate in vitro. e Addition of PAR in the demixing system promotes LLPS of the WT but not the PAR-binding deficient PBMmut hnRNP A1. f Representative images of the FRAP analysis of Alexa 647-conjugated hnRNP A1 LDs in the absence or presence of 7 μM of PAR in vitro. g The FRAP recovery curve by averaging the signals of 5~6 droplets per group with similar sizes after photobleaching. The relative fluorescence intensity of each droplet prior to photobleaching was set to 100%. Time 0 refers to the time point right after photobleaching. Mean ± SEM, ***p < 0.001; two-way ANOVA. Scale bars 10 μm in (a–e) and 2 μm in (f)

Article Snippet: In vitro liquid-liquid phase separation (LLPS) assay For the LLPS assay, purified hnRNP A1 or TDP-43 protein were mixed with PAR polymers (Trevigen, 4336–100–01) and NaCl at the indicated concentrations in a LLPS buffer (50 mM Tris-HCl, pH 7.5, 10% (w/v) PEG 3550 (Sigma) and 2mM DTT) and incubated for 3 min at room temperature.

Techniques: In Vitro, Concentration Assay, Binding Assay

Journal: Cell research

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

doi: 10.1038/s41422-019-0141-z

Figure Lengend Snippet: Fig. 5 hnRNP A1 and TDP-43 co-phase separate in vitro and PAR affects this process. a hnRNP A1 alone does not form spontaneous LDs by LLPS at the indicated condition in vitro. b–e co-LLPS experiments of hnRNP A1 with BSA (b), TDP-431–274 (c), or TDP-43274–414 (d–e) with the indicated concentrations of each protein. b′–e′ LLPS experiments of BSA (b′), TDP-431–274 (c′), or TDP-43274–414 (d′–e′) alone at the same condition as in (b–e). f Fluorophore-conjunct hnRNP A1 (Alexa Fluor-647, red) and TDP-431–247 (Alexa Fluor-555, green) confirms that the LDs formed by the co-LLPS contain both the proteins. g PAR promotes the in vitro co-LLPS of hnRNP A1 and TDP-431–274 in a dose-dependent manner. Heparin (7 μM) does not have such an effect. h PAR (7 μM) promotes the co-LLPS of hnRNP A1 (Alexa Fluor-647, red) and TDP-431–247

Article Snippet: In vitro liquid-liquid phase separation (LLPS) assay For the LLPS assay, purified hnRNP A1 or TDP-43 protein were mixed with PAR polymers (Trevigen, 4336–100–01) and NaCl at the indicated concentrations in a LLPS buffer (50 mM Tris-HCl, pH 7.5, 10% (w/v) PEG 3550 (Sigma) and 2mM DTT) and incubated for 3 min at room temperature.

Techniques: In Vitro

Journal: Cell research

Article Title: PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

doi: 10.1038/s41422-019-0141-z

Figure Lengend Snippet: Fig. 8 Downregulation of Parp or upregulation of Parg alleviates neurodegeneration in a Drosophila model of ALS. a–d Representative images (a, b) and quantifications of the degeneration scores (c, d) of the fly eyes (GMR-Gal4) expressing hTDP-43 with RNAi-Parp or RNAi-Ctrl (RNAi- mCherry) (a, c) or UAS-Parg or UAS-Ctrl (UAS-lacZ) (b, d). e, f RNAi KD of Parp in adult fly neurons using an elavGS driver (induced with RU486, 80 μg/mL, starting from day one of adulthood) alleviates the TDP-43-mediated climbing decline (e) and the lifespan shortening (f). The climbing capability is evaluated as the average percentage of flies that climb over 3 cm within 10 s. The median lifespan and the number of flies tested for each genotype are indicated. UAS-Ctrl, UAS-LacZ; RNAi-Ctrl, UAS-RNAi-mCherry. Mean ± SEM; **p < 0.01, ***p < 0.001; ns, not significant; Student’s t-test. Scale bar 100 μm. g A schematic model of the role of PARylation in regulating SGs and disease-related RNPs. hnRNPA1 is PARylated at K298 and binds to PAR and/or PARylated proteins via the PBM. PARylation and PAR-binding differentially regulate the nuclear export and SG association of hnRNPA1—the K298A mutant does not respond to stress and remains in the nucleus; the PBMmut can be recruited to SGs, but a portion of it forms mis-localized cytoplasmic foci that do not co-localize with SGs. While the formation of the RNP granules requires RNAs, the overall PARylation levels modulate the protein-protein interactions between the RBPs as well as the assembly- disassembly dynamics of the SGs. PARylation, either in the form of free PAR polymers or as PAR chains from the PARylated proteins, may act as a molecular glue to promote LLPS and enhance the assembly of SGs by cross-linking different RBPs in the granules. Pharmacological or genetic inhibition of PARP1 improves the dynamics of RNP granules and mitigates the hnRNP A1 and TDP-43 OE-mediated neurotoxicity. PAR, poly(ADP-ribose); PBM, PAR-binding motif; PPI, protein-protein interaction; WT, wild-type hnRNP A1; K298A, PARylation site mutant of hnRNP A1; PBMmut, PAR-binding motif mutant of hnRNP A1; SG, stress granule; cyto foci, abnormal cytoplasmic foci

Article Snippet: In vitro liquid-liquid phase separation (LLPS) assay For the LLPS assay, purified hnRNP A1 or TDP-43 protein were mixed with PAR polymers (Trevigen, 4336–100–01) and NaCl at the indicated concentrations in a LLPS buffer (50 mM Tris-HCl, pH 7.5, 10% (w/v) PEG 3550 (Sigma) and 2mM DTT) and incubated for 3 min at room temperature.

Techniques: Expressing, Binding Assay, Mutagenesis, Protein-Protein interactions, Inhibition