Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 4 SOX9 is the downstream gene mediated by circEHD2. A, The cluster heat maps showed the differentially expressed genes regulated by circEHD2 based on next-generation sequencing (NGS) analysis. The red and blue strips indicate up-regulated and down-regulated genes, respectively. B and C, qRT–PCR analysis of the cAMP signal pathway-related genes in OSRC-2 (B) and 786-O (C) cells with circEHD2 knockdown. D and E, qRT–PCR analysis of the expression level of SOX9 when downregulation (D) or upregulation (E) of circEHD2 in OSRC-2 and 786-O cells. F and G, Western blot showed SOX9 expression level in whole protein extracts (F) and nuclear protein extracts (G) of OSRC-2 cells when silencing circEHD2. H, Representative images of IHC and ISH showed the expression of SOX9 and circEHD2 in ccRCC tissues and paired NATs (n = 80). Scale bars: 50 μm. I, The H-score of SOX9 in ccRCC tis sues and paired NATs (n = 80). J, The Pearson correlation analysis showed that circEHD2 expression level was positively correlated with SOX9. Error bars, standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: Next-Generation Sequencing, Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Standard Deviation

Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 5 circEHD2 direct interacts with YWHAH. A, RNA pull-down assay was applied in RCC cells to identify the proteins that interacted with circEHD2. B, The silver staining image of RNA pull-down with circEHD2 probe in OSRC-2 cells. C, Mass spectrometry (MS) analysis of the proteins interacted with circEHD2 via RNA pull-down assay. D, qRT–PCR analysis confirmed that the circEHD2 probe could specifically enrich circEHD2 in OSRC-2 cells by RNA pull-down assay. E, Western blot assay in circEHD2 pull-down proteins confirmed the interaction between circEHD2 and YWHAN. F, The subcellular co- localization of circEHD2 and YWHAH in RCC cells was measured by fluorescence staining assay. Scale bars: 10 μm. G, RIP assay in OSRC-2 cells confirmed that circEHD2 could be enriched by YWHAH. IgG was used as a negative control. H, The three-dimensional structure showed circEHD2 could be bound to YWHAH. I, Serial deletions of circEHD2 were used in RNA pull-down assays to confirm the regions of circEHD2 that were required for YWHAH. J, qRT–PCR analysis of the level of circEHD2 when mutated the binding sites of circEHD2. K, RIP assay was performed in OSRC-2 cells after mutating the 201-252-nt region of circEHD2. L, The knockdown efficiency of YWHAH in RCC cells measured by qRT-PCR. M, qRT–PCR analysis of the level of SOX9 when knockdown of YWHAH in RCC cells. N, Western blot analysis of the expression of SOX9 when downregulation of YWHAH in OSRC-2 cells. O, qRT–PCR analysis of the level of SOX9 in RCC cells after mutating the 201-252-nt region of circEHD2. P, Western blot assay revealed the level of SOX9 in OSRC-2 cells after mutating the 201-252-nt region of circEHD2. Error bars, standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: Pull Down Assay, Silver Staining, Mass Spectrometry, Quantitative RT-PCR, Western Blot, Fluorescence, Staining, Negative Control, Binding Assay, Knockdown, Expressing, Standard Deviation

Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 6 YWHAH recruits circEHD2 and YWHAH to the promoter of SOX9. A and B, Co-IP assay showed that YAP could be precipitated by anti-YWHAH an tibody (A) and YWHAH could be precipitated by anti-YAP antibody (B) in OSRC-2 cells. C and D, Co-IP assay confirmed that YWHAH could bind to YAP by anti-YWHAH antibody (C) and anti-YAP antibody (D) in the nuclear extracts of OSRC-2 cells. E, Subcellular co-localization of YWHAH and YAP in RCC cells was measured by fluorescence staining assay. Scale bars: 10 μm. F, qRT–PCR analysis of the level of YAP in RCC cells when cocultured with VP at different doses. G and H, qRT–PCR analysis (G) and western blot assay (H) showed the expression level of SOX9 in OSRC-2 cells when cocultured with VP. I, Sche matic illustration of the binding sites of circEHD2 and YAP on SOX9 promoter. J, Schematic illustration of PCR-amplified fragments of SOX9 promoter. K, ChIRP analysis the enrichment of SOX9 promoter fragments in OSRC-2 cells. L, Dual-luciferase reporter assays showed that circEHD2 could bind to the P2 region (-1501-nt to -1001-nt) of the SOX9 promoter. M–P, ChIP-qPCR of YAP (M and O) and YWHAH (N and P) revealed the enrichment of SOX9 promoter in OSRC-2 cells with circEHD2 knockdown (M and N) or overexpression (O and P). Error bars represent the standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: Co-Immunoprecipitation Assay, Fluorescence, Staining, Quantitative RT-PCR, Western Blot, Expressing, Binding Assay, Amplification, Luciferase, ChIP-qPCR, Knockdown, Over Expression, Standard Deviation

Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 10 circEHD2-ASO inhibits the growth of RCC in vivo. A, Schematic illustration of tumor inoculation and circEHD2-ASO treatment in subcutaneous xenograft model and orthotopic xenograft model. B–E, Representative images of xenograft tumors (B), dissected tumors (C), the volume of tumors (D), and tumor weights (E) in subcutaneous xenograft model treated with ASO-control (n = 6/group) and circEHD2-ASO (n = 6/group), respectively. F-H, Representative images of in vivo bioluminescence imaging (F), the mean photon counts (G), and gross appearance of orthotopic tumor (H) in orthotopic xenograft model treated with ASO-control (n = 4/group) and circEHD2-ASO (n = 4/group), respectively. I, A schematic model showing the mechanism of EVs-circEHD2 mediated the progression of RCC. FUS mediated the biogenesis of circEHD2, then circEHD2 enhanced the growth of RCC through the circEHD2/YWHAH/YAP/SOX9 pathway. While hnRNPA2B1 mediated the packaging of circEHD2 into EVs, and EVs-circEHD2 promote metastasis of RCC by converting fibroblasts to CAFs. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: In Vivo, Control, Imaging

Journal: Theranostics

Article Title: Vitamin D binding protein (VDBP) hijacks twist1 to inhibit vasculogenic mimicry in hepatocellular carcinoma

doi: 10.7150/thno.90322

Figure Lengend Snippet: Materials

Article Snippet: Mouse monoclonal anti-Vitamin D binding protein (DBP) , , Proteintech , CAT#66175-1-Ig , RRID: AB_2881570.

Techniques: Luciferase, Reporter Gene Assay, Silver Staining, Binding Assay, Molecular Weight, In Situ, Membrane, Magnetic Beads, Transfection, Modification, H&E Stain, Staining, cDNA Synthesis, SYBR Green Assay, Bicinchoninic Acid Protein Assay, In Vivo, Plasmid Preparation, CRISPR, Knock-Out, Sequencing, Software, Imaging

Journal: Scientific Reports

Article Title: ADAMTS-13 regulates neutrophil recruitment in a mouse model of invasive pulmonary aspergillosis

doi: 10.1038/s41598-017-07340-3

Figure Lengend Snippet: A . fumigatus infection of Adamts13 −/− mice results in increased mortality and fungal burden of the lungs. Wildtype (129\Sv\Pas) and Adamts13 −/− mice were infected i . t . with A . fumigatus (10 7 conidia per animal). ( A ) Overall survival were monitored for 14 days. The cumulative results of three independent experiments (129/Sv/Pas, n = 15; 129/Sv/Pas + Gr-1, n = 10; Adamts13 −/− , n = 17) are depicted. (*) indicates a significant difference (p < 0.05) by Mantel-Cox-Test. ( B ) 24 h after infection, some mice were sacrificed and their lungs were prepared and homogenized. The fungal burden was determined by plating serial dilutions of lung homogenates on Sabouraud 4% glucose agar plates. After 48 h the resulting colony-forming units (CFU) were enumerated. Shown are the cumulative results of three independent experiments (129/Sv/Pas, n = 10; Adamts13 −/− , n = 11) plus SD. (*) indicates a significant difference (p < 0.05) by Mann-Whitney U-test. ( C ) The albumin concentration of the BAL fluid was quantified by ELISA 24 h after infection. The cumulative results of two (H 2 O-treated 129/Sv/Pas, n = 6), three ( A . fumigatus -treated Adamts13 −/− , n = 11) or four independent experiments ( A . fumigatus -treated 129/Sv/Pas, n = 13) plus SD are depicted. ( D ) Plasma LDH activity was measured 24 h after infection of wildtype (n = 4) and Adamts13 −/− (n = 5) mice. ( E – H ) 24 h after infection, three mice of the indicated groups were sacrificed, paraffin sections of the lungs were prepared and stained with H&E, Grocott ( F ), C3d antibody ( G ) and VWF antibody ( H ). ( E ) Perivascular inflammation were scored by S. R. who was blinded to the experimental treatment groups. (*) indicates a significant difference (p < 0.05) by one-way ANOVA with Bonferroni’s posttest. ( F – H ) Representative images of lung sections are depicted (H&E, Grocott and C3d 400x magnification, VWF 800x magnification), vessels are marked by an arrow.

Article Snippet: Paraffin embedded lung sections were stained with hematoxylin and eosin (H&E) to assess inflammatory responses, Grocott-Gomori silver stain to visualize fungi, mouse complement component C3d antibody (R&D systems) and VWF antibody (Dako).

Techniques: Infection, MANN-WHITNEY, Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Activity Assay, Staining

Journal: Cell reports

Article Title: Intracellular complement C5a/C5aR1 stabilizes β-catenin to promote colorectal tumorigenesis.

doi: 10.1016/j.celrep.2022.110851

Figure Lengend Snippet: Figure 6. CTSD cleaves C5 into C5a in CRC (A) RNA-seq analysis of the mRNA levels of various proteases, including Ctsd (red), in colonic tumors or the counterpart tissues of mice with or without AOM and DSS treatment, respectively. Data are represented as RPKM value for the 23 upregulated protease genes. (B) Kaplan-Meier analysis of the association between overall survival and the mRNA level of CTSD in tumor tissues of CRC patients (n = 364). Data were from The Cancer Genome Atlas (TCGA) database. (C) CTSD cleaved C5 to produce C5a in a dose-dependent manner, detected by Coomassie staining. (D and E) The mRNA (D) and protein (E) levels of CTSD in colons of AOM and DSS-treated or control mice detected by real-time PCR or immunoblotting assay, respectively. Data are represented as mean ± SEM; n R 4; **p < 0.01. (F) The local levels of mC5a in colonic tissue homogenates (CTHs) of mice with or without AOM and DSS treatment measured by ELISA. Data are represented as mean ± SEM; n R 4; *p < 0.05. (G) The effect of CTSD insufficiency on the intracellular C5a levels measured by ELISA. Data are represented as mean ± SD; n = 3; *p < 0.05; ***p < 0.001. (H) The effect of CTSD insufficiency on b-catenin protein levels. (I) A proposed model underlying the mechanism of intracellular C5a/C5aR1 signaling in colonic epithelial cells for regulating b-catenin stability via assembling a b-catenin stabilization complex of C5a/C5aR1/KCTD5/cullin3/Roc-1/b-catenin. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PMX205 R&D Systems Cat#5196/1 Cycloheximide (CHX) CST Cat#2112 recombinant human cathepsin D protein R&D Systems Cat# 1014-AS-010 recombinant human KCTD5 protein (His Tag) Abcam Cat#ab137135 beta-Catenin Protein, Human, Recombinant (GST Tag) Sino Biological Cat#11279-H20B C5a Anaphylatoxin CompTech Cat#A144 C5 CompTech Cat#A120 Azoxymethane (AOM) Sigma-Aldrich Cat#A5486 Dextran Sulfate Sodium Salt (DSS 36,000–50,000 M.Wt) MP Biomedical CAS:9011-18-1 Critical commercial assays Human Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2037 Mouse Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2150 GTVision III immunohistochemical detection kit Gene Tech Cat#GK500705 KOD-Plus-Mutagenesis Kit TOYOBO Cat#SMK-101 Fast Silver Stain Kit Beyotime Cat#P0017S MinuteTM Cytoplasmic and Nuclear Fractionation kit Invent Biotechnologies Cat#SC-003 MinuteTM Endosome Isolation and Cell Fractionation Kit Invent Biotechnologies Cat#ED-028 Human Neutrophil Isolation Kit TBD Cat#LZS11131 TMB Substrate Set Biolegend Cat#421101 Stop Solution for TMB Substrate Biolegend Cat#423001 DAPI Fluoromount-G YEASEN Cat#36308ES11 Fixation Buffer Biolegend Cat#420801 Permeabilization Wash Buffer (10X) Biolegend Cat#421002 Protein A/G Magnetic Beads Bimake Cat#B23202 PrimeScriptTM RT Master Mix Takara Cat#RR036A TB Green Premix Ex TaqTM II Takara Cat#RR820A Deposited data RNA sequencing data NCBI GEO database GEO#GSE127946 4D-label free quantitative proteomics data This study See Tables S1 and S2 Protein profiling by LC-MS This study See Table S3 Experimental models: Cell lines HT-29 ATCC Cat#HTB-38 RKO ATCC Cat#CRL-2577 SW480 ATCC Cat#CCL-228 SW620 ATCC Cat#CCL-227 LoVo ATCC Cat#CCL-229 DLD-1 ATCC Cat#CCL-221 HCT 116 ATCC Cat#CCL-247 HCT-8 ATCC Cat#CCL-244 HCT-15 ATCC Cat#CCL-225 NCM460 Professor Guoxiang Cai N/A HCoEpiC Jennio-Bio N/A CT26.WT ATCC Cat#CRL-2638 HEK293FT Thermo Fisher Cat#R70007 HepG2 ATCC Cat#HB-8065 Experimental models: Organisms/strains C57BL/6 mice SLAC Laboratory Animal Co., Ltd N/A B10.D2-Hc0 mice The Jackson Laboratory Cat#000461 (Continued on next page) Cell Reports 39, 110851, May 31, 2022 e2

Techniques: RNA Sequencing, Staining, Control, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Intracellular complement C5a/C5aR1 stabilizes β-catenin to promote colorectal tumorigenesis.

doi: 10.1016/j.celrep.2022.110851

Figure Lengend Snippet: Figure 7. Correlation of C5a, C5aR1, b-catenin, and CTSD in human CRC specimens (A and B) Representative images of C5a, C5aR1, b-catenin, and CTSD staining in normal colon, adenoma, and adenocarcinoma tissues in clinical CRC spec- imens (A) or in paired CRC adenocarcinoma tissues and adjacent non-tumoral tissues (B). Scale bars, inner, 200 mm; outer, 50 mm. (C and D) Statistical analysis of the histological staining score (H score) in CRC specimens. Data are represented as min to max in (C) (related to A); n = 16 (normal), 35 (adenoma), or 9 (adenocarcinoma). Data of nine paired adenocarcinoma and adjacent tissues are shown in (D) (related to B); ns, not significant;***p < 0.001; ****p < 0.0001. (E and F) Scatter diagram analysis showing highly positive correlations of b-catenin with C5a or C5aR1 (E) or of CTSD with C5a or b-catenin (F).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PMX205 R&D Systems Cat#5196/1 Cycloheximide (CHX) CST Cat#2112 recombinant human cathepsin D protein R&D Systems Cat# 1014-AS-010 recombinant human KCTD5 protein (His Tag) Abcam Cat#ab137135 beta-Catenin Protein, Human, Recombinant (GST Tag) Sino Biological Cat#11279-H20B C5a Anaphylatoxin CompTech Cat#A144 C5 CompTech Cat#A120 Azoxymethane (AOM) Sigma-Aldrich Cat#A5486 Dextran Sulfate Sodium Salt (DSS 36,000–50,000 M.Wt) MP Biomedical CAS:9011-18-1 Critical commercial assays Human Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2037 Mouse Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2150 GTVision III immunohistochemical detection kit Gene Tech Cat#GK500705 KOD-Plus-Mutagenesis Kit TOYOBO Cat#SMK-101 Fast Silver Stain Kit Beyotime Cat#P0017S MinuteTM Cytoplasmic and Nuclear Fractionation kit Invent Biotechnologies Cat#SC-003 MinuteTM Endosome Isolation and Cell Fractionation Kit Invent Biotechnologies Cat#ED-028 Human Neutrophil Isolation Kit TBD Cat#LZS11131 TMB Substrate Set Biolegend Cat#421101 Stop Solution for TMB Substrate Biolegend Cat#423001 DAPI Fluoromount-G YEASEN Cat#36308ES11 Fixation Buffer Biolegend Cat#420801 Permeabilization Wash Buffer (10X) Biolegend Cat#421002 Protein A/G Magnetic Beads Bimake Cat#B23202 PrimeScriptTM RT Master Mix Takara Cat#RR036A TB Green Premix Ex TaqTM II Takara Cat#RR820A Deposited data RNA sequencing data NCBI GEO database GEO#GSE127946 4D-label free quantitative proteomics data This study See Tables S1 and S2 Protein profiling by LC-MS This study See Table S3 Experimental models: Cell lines HT-29 ATCC Cat#HTB-38 RKO ATCC Cat#CRL-2577 SW480 ATCC Cat#CCL-228 SW620 ATCC Cat#CCL-227 LoVo ATCC Cat#CCL-229 DLD-1 ATCC Cat#CCL-221 HCT 116 ATCC Cat#CCL-247 HCT-8 ATCC Cat#CCL-244 HCT-15 ATCC Cat#CCL-225 NCM460 Professor Guoxiang Cai N/A HCoEpiC Jennio-Bio N/A CT26.WT ATCC Cat#CRL-2638 HEK293FT Thermo Fisher Cat#R70007 HepG2 ATCC Cat#HB-8065 Experimental models: Organisms/strains C57BL/6 mice SLAC Laboratory Animal Co., Ltd N/A B10.D2-Hc0 mice The Jackson Laboratory Cat#000461 (Continued on next page) Cell Reports 39, 110851, May 31, 2022 e2

Techniques: Staining

Journal: iScience

Article Title: PABPC1 mediates degradation of C9orf72 -FTLD/ALS GGGGCC repeat RNA

doi: 10.1016/j.isci.2024.109303

Figure Lengend Snippet:

Article Snippet: Rabbit anti-EXOSC10 (WB: 1:500, PLA: 1:100) , Atlas Antibodies , Cat# HPA028484; RRID: AB_10611250.

Techniques: FLAG-tag, Virus, Recombinant, Magnetic Beads, Plasmid Preparation, Protease Inhibitor, Blocking Assay, Reverse Transcription, Silver Staining, In Situ, Expressing, Construct, Software, Molecular Weight, Laser-Scanning Microscopy, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: C5a increased in children with idiopathic nephrotic syndrome when the disease flared up. a A representative cytokine array data of the paired sera samples from a patient during relapse and remission was presented. Marked area denotes the duplicated spots where C5a antibody was dotted. b The pixel density of each duplicated C5a spot was measured and the average calculated. The ratio of the C5a spots intensity for paired samples during relapse and remission from four patients were presented. c Serum C5a changes from a cohort of 22 children with idiopathic nephrotic syndrome during relapse and remission were determined by ELISA. The result shows that C5a level significantly increased in this population when nephrotic syndrome was manifested (C5a = 44.30 ± 32.95 ng/ml during relapse vs. 22.31 ± 14.62 ng/ml during remission; mean ± SD, N = 22. **P = 0.005). d Sera were collected from 5 children afflicted with nephrotic syndrome whose serum C5a values were assessed to be the highest among the cohort as discussed in (c) during the relapse stage. Then undiluted sera (filled square, relapse; open square, remission) from these five children were each incubated with HUVECs for 60 min to perform the permeability assays as described in the “Materials and methods” section. In a tested group of Transwells, the assays were performed in the presence of C5a receptor antagonist W-54011 (10 nM). Serum samples which had been heated at 56 °C for 30 min served as negative controls. The data shown represent mean ± SD. *P < 0.05

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Permeability

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: C5a-induced permeability change towards protein in kidney endothelial cells. Kidney endothelial cells (KECs) and glomerular podocytes were isolated by antibody-coated magnetic beads form mouse kidney tissue as described in the “Materials and methods” section. a The isolated KECs (E) and podocytes (P) were further processed by semi-quantitative RT-PCR (mRNA) and Western blotting analysis (protein) using the indicated markers to confirm the purity of each cellular population. b The homogeneity of the KECs was further verified by immunostaining with endothelial cell-specific surface marker CD31 followed by flow cytometry (empty shadow as a negative control in which isotype specific control antibody was added). The percentage of endothelial cells was quantified to be 96.56 % positive for CD31. c Phase contrast image of KECs with ×200 magnifications. Scale bars 10 μm. d KECs were cultured on Transwell plates and treated with the indicated dosages of C5a for 60 min before permeability assays were performed as described in “Materials and methods” section. The data shown represent mean ± SD (N = 5). *P < 0.05; **P < 0.001

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Permeability, Isolation, Magnetic Beads, Quantitative RT-PCR, Western Blot, Immunostaining, Marker, Flow Cytometry, Negative Control, Control, Cell Culture

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: C5a elicited nephrotic syndrome like manifestation in mice. ICR mice were injected with 200 ng recombinant C5a (10 μg/kg) every 3 days through tail vein. Urinary albumin/creatinine (A/C) ratio (a), serum albumin (b), triglyceride (c), and total cholesterol (d) levels were monitored every other day in the experimental group (filled square) and control mice which were injected with normal saline (open square). N = 5 for each group. The data shown represent mean ± SD. *P < 0.05; **P < 0.001

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Injection, Recombinant, Control, Saline

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: The pathological analysis of kidney tissues from C5a-treated mice manifested a pattern compatible with minimal change disease. ICR mice were treated with 200 ng recombinant C5a (10 μg/kg) every 3 days. Kidney tissues from mice killed on day 0, 6, 15 and 21 were dissected and processed for H&E (a), PAS (b), Roussel-Silver staining (c), and electron microscopic examination (d) in which mild focal effacement of some podocytes was noted (asterisk sign). Scale bars 10 μm for panels a, b, and c; scale bars 0.5 μm for panel d

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Recombinant, Silver Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: C5a-induced disassembly and redistribution of adherens junctional proteins. Mouse KECs were grown to confluency. The culture medium was replaced with M199 medium for serum fasting overnight before the indicated concentration of C5a (ng/ml) was applied to the cells for 60 min. After phase contrast images of KECs were acquired with CCD camera (a), the KECs were processed for immunofluorescence using mouse anti-VE cadherin (b) and rabbit anti-β-catenin (c) antibodies. The actin cytoskeleton organization was revealed by TRITC-rhodamine labeled phalloidin to stain for polymerized actin (d). Scale bars 10 μm

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Concentration Assay, Immunofluorescence, Labeling, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: C5a-induced cellular contraction, myosin contraction and actin stress fiber formation of KECs in a ROCK dependent way. a KECs were serum fasted overnight in M199 medium before C5a (50 ng/ml) was applied to the cells for 60 min with (right) or without (left) 10 μM Y27632 pretreatment for 30 min before phase contrast images were taken. b The culture condition was the same as in (a) except the cells were maintained at a confluent density before staining with non-muscle myosin II heavy chain B antibody (MYHb, red) and FITC-labeled phalloidin (green). c C5a effect on the actin cytoskeleton in vivo was shown. The intensive phalloidin staining in microvilli of the surrounding renal tubules (arrowheads) serves as a reference point to compare the intensities of phalloidin staining in the glomeruli (arrows) on fresh frozen renal sections from control or C5a (50 ng/ml) treated mice. d Confocal sectioning result following immunofluorescence study using VE-cadherin labeling on fresh frozen renal sections from control or C5a (50 ng/ml) treated mice was shown. Scale bars 10 μm

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Staining, Labeling, In Vivo, Control, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: C5a-induced RhoA/ROCK1-dependent myosin light chain activation which led to an increase in the permeability of KECs. KECs were serum fasted in M199 medium for 3 h. Cells were further incubated for 60 min in the presence of the indicated concentration of recombinant C5a before cell lysates were harvested for Rho activation assay (a), Western blotting analysis using the antibodies recognizing total and phospho-specific ROCK1 (b), and total myosin light chain (MLC) and phosphoserine-specific MLC at the amino acid position 19 (c). Y, 10 μM Y27632; PTX, 100 ng/ml pertussis toxin pre-treated for 30 min. (d) Renal glomeruli were isolated from mice treated with 50 ng/ml recombinant C5a as the procedure described in the “Materials and methods” when the proteinuria was first evident. Cell lysates were prepared in NP-40 buffer for RhoA activation assay. e, f KECs were cultured on Transwell plates and pretreated with the indicated inhibitors for 30 min. Then, 50 ng/ml recombinant C5a was applied into the transwell plates for an additional 60 min before permeability assays were performed. The data shown represent mean ± SD (N = 4). *P < 0.05; **P < 0.001

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Activation Assay, Permeability, Incubation, Concentration Assay, Recombinant, Western Blot, Isolation, Cell Culture

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: Y27632 inhibited the C5a-elicited proteinuria in a murine model. ICR mice were injected with 200 ng recombinant C5a (10 μg/kg) every 3 days through tail vein for 21 days, and Y27632 were administrated 0.6 mg (30 mg/kg) orally once daily since day 6. Urinary albumin/creatinine ratio (a), serum albumin (b), triglyceride (c), and total cholesterol (d) levels were monitored every 3 days. The data shown represent mean ± SD for each treatment group (N = 5). *P < 0.05

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 μg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Injection, Recombinant

Journal: Cell reports

Article Title: Intracellular complement C5a/C5aR1 stabilizes β-catenin to promote colorectal tumorigenesis.

doi: 10.1016/j.celrep.2022.110851

Figure Lengend Snippet: Figure 6. CTSD cleaves C5 into C5a in CRC (A) RNA-seq analysis of the mRNA levels of various proteases, including Ctsd (red), in colonic tumors or the counterpart tissues of mice with or without AOM and DSS treatment, respectively. Data are represented as RPKM value for the 23 upregulated protease genes. (B) Kaplan-Meier analysis of the association between overall survival and the mRNA level of CTSD in tumor tissues of CRC patients (n = 364). Data were from The Cancer Genome Atlas (TCGA) database. (C) CTSD cleaved C5 to produce C5a in a dose-dependent manner, detected by Coomassie staining. (D and E) The mRNA (D) and protein (E) levels of CTSD in colons of AOM and DSS-treated or control mice detected by real-time PCR or immunoblotting assay, respectively. Data are represented as mean ± SEM; n R 4; **p < 0.01. (F) The local levels of mC5a in colonic tissue homogenates (CTHs) of mice with or without AOM and DSS treatment measured by ELISA. Data are represented as mean ± SEM; n R 4; *p < 0.05. (G) The effect of CTSD insufficiency on the intracellular C5a levels measured by ELISA. Data are represented as mean ± SD; n = 3; *p < 0.05; ***p < 0.001. (H) The effect of CTSD insufficiency on b-catenin protein levels. (I) A proposed model underlying the mechanism of intracellular C5a/C5aR1 signaling in colonic epithelial cells for regulating b-catenin stability via assembling a b-catenin stabilization complex of C5a/C5aR1/KCTD5/cullin3/Roc-1/b-catenin. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PMX205 R&D Systems Cat#5196/1 Cycloheximide (CHX) CST Cat#2112 recombinant human cathepsin D protein R&D Systems Cat# 1014-AS-010 recombinant human KCTD5 protein (His Tag) Abcam Cat#ab137135 beta-Catenin Protein, Human, Recombinant (GST Tag) Sino Biological Cat#11279-H20B C5a Anaphylatoxin CompTech Cat#A144 C5 CompTech Cat#A120 Azoxymethane (AOM) Sigma-Aldrich Cat#A5486 Dextran Sulfate Sodium Salt (DSS 36,000–50,000 M.Wt) MP Biomedical CAS:9011-18-1 Critical commercial assays Human Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2037 Mouse Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2150 GTVision III immunohistochemical detection kit Gene Tech Cat#GK500705 KOD-Plus-Mutagenesis Kit TOYOBO Cat#SMK-101 Fast Silver Stain Kit Beyotime Cat#P0017S MinuteTM Cytoplasmic and Nuclear Fractionation kit Invent Biotechnologies Cat#SC-003 MinuteTM Endosome Isolation and Cell Fractionation Kit Invent Biotechnologies Cat#ED-028 Human Neutrophil Isolation Kit TBD Cat#LZS11131 TMB Substrate Set Biolegend Cat#421101 Stop Solution for TMB Substrate Biolegend Cat#423001 DAPI Fluoromount-G YEASEN Cat#36308ES11 Fixation Buffer Biolegend Cat#420801 Permeabilization Wash Buffer (10X) Biolegend Cat#421002 Protein A/G Magnetic Beads Bimake Cat#B23202 PrimeScriptTM RT Master Mix Takara Cat#RR036A TB Green Premix Ex TaqTM II Takara Cat#RR820A Deposited data RNA sequencing data NCBI GEO database GEO#GSE127946 4D-label free quantitative proteomics data This study See Tables S1 and S2 Protein profiling by LC-MS This study See Table S3 Experimental models: Cell lines HT-29 ATCC Cat#HTB-38 RKO ATCC Cat#CRL-2577 SW480 ATCC Cat#CCL-228 SW620 ATCC Cat#CCL-227 LoVo ATCC Cat#CCL-229 DLD-1 ATCC Cat#CCL-221 HCT 116 ATCC Cat#CCL-247 HCT-8 ATCC Cat#CCL-244 HCT-15 ATCC Cat#CCL-225 NCM460 Professor Guoxiang Cai N/A HCoEpiC Jennio-Bio N/A CT26.WT ATCC Cat#CRL-2638 HEK293FT Thermo Fisher Cat#R70007 HepG2 ATCC Cat#HB-8065 Experimental models: Organisms/strains C57BL/6 mice SLAC Laboratory Animal Co., Ltd N/A B10.D2-Hc0 mice The Jackson Laboratory Cat#000461 (Continued on next page) Cell Reports 39, 110851, May 31, 2022 e2

Techniques: RNA Sequencing, Staining, Control, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Intracellular complement C5a/C5aR1 stabilizes β-catenin to promote colorectal tumorigenesis.

doi: 10.1016/j.celrep.2022.110851

Figure Lengend Snippet: Figure 7. Correlation of C5a, C5aR1, b-catenin, and CTSD in human CRC specimens (A and B) Representative images of C5a, C5aR1, b-catenin, and CTSD staining in normal colon, adenoma, and adenocarcinoma tissues in clinical CRC spec- imens (A) or in paired CRC adenocarcinoma tissues and adjacent non-tumoral tissues (B). Scale bars, inner, 200 mm; outer, 50 mm. (C and D) Statistical analysis of the histological staining score (H score) in CRC specimens. Data are represented as min to max in (C) (related to A); n = 16 (normal), 35 (adenoma), or 9 (adenocarcinoma). Data of nine paired adenocarcinoma and adjacent tissues are shown in (D) (related to B); ns, not significant;***p < 0.001; ****p < 0.0001. (E and F) Scatter diagram analysis showing highly positive correlations of b-catenin with C5a or C5aR1 (E) or of CTSD with C5a or b-catenin (F).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PMX205 R&D Systems Cat#5196/1 Cycloheximide (CHX) CST Cat#2112 recombinant human cathepsin D protein R&D Systems Cat# 1014-AS-010 recombinant human KCTD5 protein (His Tag) Abcam Cat#ab137135 beta-Catenin Protein, Human, Recombinant (GST Tag) Sino Biological Cat#11279-H20B C5a Anaphylatoxin CompTech Cat#A144 C5 CompTech Cat#A120 Azoxymethane (AOM) Sigma-Aldrich Cat#A5486 Dextran Sulfate Sodium Salt (DSS 36,000–50,000 M.Wt) MP Biomedical CAS:9011-18-1 Critical commercial assays Human Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2037 Mouse Complement Component C5a DuoSet ELISA kit R&D Systems Cat#DY2150 GTVision III immunohistochemical detection kit Gene Tech Cat#GK500705 KOD-Plus-Mutagenesis Kit TOYOBO Cat#SMK-101 Fast Silver Stain Kit Beyotime Cat#P0017S MinuteTM Cytoplasmic and Nuclear Fractionation kit Invent Biotechnologies Cat#SC-003 MinuteTM Endosome Isolation and Cell Fractionation Kit Invent Biotechnologies Cat#ED-028 Human Neutrophil Isolation Kit TBD Cat#LZS11131 TMB Substrate Set Biolegend Cat#421101 Stop Solution for TMB Substrate Biolegend Cat#423001 DAPI Fluoromount-G YEASEN Cat#36308ES11 Fixation Buffer Biolegend Cat#420801 Permeabilization Wash Buffer (10X) Biolegend Cat#421002 Protein A/G Magnetic Beads Bimake Cat#B23202 PrimeScriptTM RT Master Mix Takara Cat#RR036A TB Green Premix Ex TaqTM II Takara Cat#RR820A Deposited data RNA sequencing data NCBI GEO database GEO#GSE127946 4D-label free quantitative proteomics data This study See Tables S1 and S2 Protein profiling by LC-MS This study See Table S3 Experimental models: Cell lines HT-29 ATCC Cat#HTB-38 RKO ATCC Cat#CRL-2577 SW480 ATCC Cat#CCL-228 SW620 ATCC Cat#CCL-227 LoVo ATCC Cat#CCL-229 DLD-1 ATCC Cat#CCL-221 HCT 116 ATCC Cat#CCL-247 HCT-8 ATCC Cat#CCL-244 HCT-15 ATCC Cat#CCL-225 NCM460 Professor Guoxiang Cai N/A HCoEpiC Jennio-Bio N/A CT26.WT ATCC Cat#CRL-2638 HEK293FT Thermo Fisher Cat#R70007 HepG2 ATCC Cat#HB-8065 Experimental models: Organisms/strains C57BL/6 mice SLAC Laboratory Animal Co., Ltd N/A B10.D2-Hc0 mice The Jackson Laboratory Cat#000461 (Continued on next page) Cell Reports 39, 110851, May 31, 2022 e2

Techniques: Staining