Journal: bioRxiv

Article Title: The SUMO ligase Su(var)2-10 links piRNA-guided target recognition to chromatin silencing

doi: 10.1101/533091

Figure Lengend Snippet: (A)Schematic diagram of the reporter used to study the effect of Su(var)2-10 recruitment to RNA. GFP-Su(var)2-10 or just GFP (control) are fused to the RNA-biding AN domain, which has high affinity for BoxB hairpins and the mKate reporter encoding 4 BoxB hairpins in the 3’UTR are co-expressed in ovaries resulting in Su(var)2-10 recruitment to the reporter’s nascent transcript. Regions A-C denote the positions of RT-qPCR amplicons. (B)Tethering of Su(var)2-10 leads to reduced reporter mRNA level. Boxplot shows reporter expression levels (region B) in ovaries expressing AN-GFP-Su(var)2-10 or AN-GFP control as estimated by RT-qPCR. Error bars show the standard deviation from 3 biological replicates. Statistical significance is estimated by two-tailed Student’s t-test. ***p<0.001 (C)Tethering of Su(var)2-10 leads to decreased Pol II occupancy at the reporter locus. Boxplot shows Pol II occupancy at the reporter locus in ovaries expressing AN-GFP-Su(var)2-10 or AN-GFP control as estimated by ChIP-qPCR. Data is normalized to Rp49 as endogenous control, and fold enrichment is calculated as the ratio of signal in ChIP to signal in input. Error bars show the standard deviation from two biological replicates. Statistical significance is estimated by two-tailed Student’s t-test. *p<0.05 (D)Tethering of Su(var)2-10 leads to H3K9me3 deposition at reporter locus. Boxplot shows H3K9me3 levels at the reporter locus in ovaries expressing AN-GFP-Su(var)2-10 or AN-GFP control as estimated by ChIP-qPCR (for regions A-C). Data is normalized to Rp49 as endogenous control, and fold enrichment is calculated as the ratio of signal in ChIP to signal in input. Error bars show the standard deviation from two biological replicates. Statistical significance is estimated by two-tailed Student’s t-test. *p<0.05 (E)Schematic diagram of Drosophila Su(var)2-10 protein structure (PA isoform). Grey boxes mark conserved domains. Local alignment of the SP-RING domain between human PIAS1 and Su(var)2-10 is shown, highlighting the catalytic cysteine residue identified in human PIAS1. Sequences were extracted from UniProt and aligned by MUSCLE . (F)Su(var)2-10 interacts with SUMO. S2 cell lysates expressing GFP-Su(var)2-10 were incubated with bacterially expressed GST-SUMO (wild type), interaction-deficient mutant GST-SUMO (QFI>AAA), or no bait control. GST-SUMO was affinity purified using glutathione sepharose beads and GFP-Su(var)2-10 was detected by Western blotting using an anti-GFP antibody. (G)Su(var)2-10 interacts with Ubc9. S2 cell lysates expressing FLAG-tagged Ubc9 and GFP-Su(var)2-10 were analysed by immunoprecipitation using GFP nanotrap beads. FLAG-Ubc9 co-purifies with GFP-Su(var)2-10. (H)Su(var)2-10 tethering promotes SUMO enrichment at the reporter locus. Boxplot shows SUMO levels at reporter locus in ovaries expressing AN-GFP-Su(var)2-10 (wild type or mutant) or AN-GFP control as estimated by ChIP-qPCR (region A). Wild type but not C341S Su(var)2-10 mutant induces SUMO enrichment. Data is normalized to Rp49 as endogenous control, and fold enrichment is calculated as the ratio of signal in ChIP to signal in input. Error bars show the standard deviation from two biological replicates. Statistical significance is estimated by one-way ANOVA followed by Tukey’s test; *p<0.05. (I)Su(var)2-10 mutant (C341S) is unable to repress reporter transcription. AN-GFP control, AN-GFP-Su(var)2-10 (wild type) or AN-GFP-Su(var)2-10 C341S mutant were co-expressed in ovaries with mKate-4BoxB reporter. Bar plots show reporter expression levels (region B) measured by RT-qPCR. Tethering of wild type, but not catalytically inactive AN-GFP-Su(var)2-10 induces reporter repression. Error bars show the standard deviation of three biological replicates. Statistical significance is estimated by one-way ANOVA followed by Tukey’s test; **p<0.01. (J) Catalytically inactive Su(var)2-10 mutant (C341S) is unable to induce H3K9me3 deposition. Bar plots show H3K9me3 levels at the mKate-4BoxB reporter upon tethering of AN-GFP control, AN-GFP-Su(var)2-10 (wild type) or AN-GFP-Su(var)2-10 C341S as measured by ChIP-qPCR (region B). Recruitment of wild type, but not catalytically inactive AN-GFP-Su(var)2-10 induces H3K9me3 enrichment. ChIP data is analysed as in D. Error bars show standard deviation of two biological replicates. Statistical significance is estimated by one-way ANOVA followed by Tukey’s test; *p<0.05.

Article Snippet: ChIPs were carried out as described previously( ) with the following antibodies: anti-H3K9me3 [ab8898], anti-RNA Pol II [ab5408], anti-H3K4me2/3 [Ab6000], anti-H3K36me3 [ab9050], HP1a [C1A9, DSHB] and anti-Drosophila SUMO (smt3), a kind gift from G Cavalli( ).

Techniques: Control, Quantitative RT-PCR, Expressing, Standard Deviation, Two Tailed Test, ChIP-qPCR, Residue, Incubation, Mutagenesis, Affinity Purification, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: A brain-enriched Drp1 isoform associates with lysosomes, late endosomes, and the plasma membrane

doi: 10.1074/jbc.RA117.001253

Figure Lengend Snippet: Drp1ABCD is located at interfaces between mitochondria and lysosomes/late endosomes. A, Drp1-KO MEFs carrying Drp1ABCD and S-Drp1 were analyzed by immunofluorescence microscopy with antibodies to Lamp1, Drp1, and the mitochondrial protein Tom20. A gallery of Lamp1-positive vesicles is shown. Three examples for each Drp1 construct are shown. Scale bar = 2 μm. B, quantification of Lamp1-positive vesicles that are in close contact with mitochondria in Drp1-KO MEFs expressing Drp1ABCD. We measured the frequency of Lamp1-positive membranes that are closely faced to mitochondria when Drp1ABCD is present (+) or not (−), as depicted in the schematic. Error bars represent the average ± S.D. (n = 61). Statistical analysis was performed using Student's t test: ***, p < 0.001. C, line scan analysis of the fluorescent signal of Lamp1, Drp1ABCD, and Tom20 in immunofluorescence microscopy of Drp1-KO MEFs expressing Drp1ABCD. Scale bar = 1 μm. The fluorescent intensity was quantified along the dotted line. The peak position of Drp1 is located between those of Lamp1 and Tom20. D, mitochondria were isolated from Drp1-KO MEFs expressing S-Drp1 or Drp1ABCD using a mitochondrial isolation kit (Thermo, 89874). Whole-cell lysates and mitochondrial fractions were analyzed by Western blotting with antibodies against Drp1, Lamp1, ATP6V1C1, Tom20, and GAPDH.

Article Snippet: The antibodies used were exon AB, Drp1 (BD Biosciences, 611113), Tom20 (BD Biosciences, 612278), PDH subunit E2/E3bp (Abcam, ab110333), GAPDH (Thermo, MA5-15738), actin (Santa Cruz Biotechnology, sc-1615), Lamp1 (BD Biosciences, 553792), ATP6V1C1 (ProteinTech, 16054-1-AP), and Rab7 (Cell Signaling Technology, 9367).

Techniques: Immunofluorescence, Microscopy, Construct, Expressing, Isolation, Western Blot

Journal: Molecular Medicine Reports

Article Title: Proteomics analysis of lung tissue reveals protein makers for the lung injury of adjuvant arthritis rats

doi: 10.3892/mmr.2023.13051

Figure Lengend Snippet: Immunofluorescence results for C1qc, C9 and Clu (×400). (A) Immunofluorescence expression of C1qc. (B) Immunofluorescence expression of C9. (C) Immunofluorescence expression of Clu. Quantitative analysis of immunofluorescence results of (D) C1qc, (E) C9 and (F) Clu proteins. Relative expression of (G) C1qc, (H) C9 and (I) Clu proteins were analyzed by proteomic analysis. *P<0.05 or **P<0.01 vs. CN group in rats. C, complement; CN, control; Clu, clusterin.

Article Snippet: The sections were incubated with primary antibodies against C1qc (1:100; cat. no. A05666; Boster Biological Technology), C9 (1:100; cat. no. K107807P; Beijing Solarbio Science and Technology Co., Ltd.), Clu (1:100; cat. no. 12289-1-AP; Proteintech Group, Inc.) at 4°C overnight and washed with PBS 3 times (5 min each).

Techniques: Immunofluorescence, Expressing, Control

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: KTN1-AS1 interacts with RBBP4 in the nucleus. ( A ) The subcellular location of KTN1-AS1 in esophageal squamous cell carcinoma (ESCC) cell lines. ( B ) RNA pull-down assay was performed in Kyse150 and Kyse170 cells, and the RNA-related proteins were determined with SDS-PAGE gel and coomassie brilliant blue staining. Original gel image was presented in Supplementary Fig. A. ( C ) Western blot assay was performed to detect the specific association between RBBP4 and KTN1-AS1 in Kyse150 and Kyse170 cells. Original western blots were presented in Supplementary Fig. B, with blots cut prior to hybridization with antibodies. ( D ) RIP assay showed the interaction between KTN1-AS1 and RBBP4 in Kyse150 and Kyse170 cells. ( E ) The regulation effect of KTN1-AS1 on RBBP4 expression was detected by qRT-PCR and western blot. Original western blots were presented in Supplementary Fig. C, with blots cut prior to hybridization with antibodies. ( F ) The relevance between KTN1-AS1 and RBBP4 expression was predicted by the GEPIA database. ( G ) The relative expression of RBBP4 in 182 tumor samples compared with 286 normal samples obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) database. ( H ) The expression levels of RBBP4 in ESCC and the corresponding normal tissues. ( I ) The expression levels of RBBP4 in ESCC cell lines. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Pull Down Assay, SDS Page, Staining, Western Blot, Hybridization, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: KTN1-AS1 relates to epithelial-to-mesenchymal transition (EMT) process by interacting with RBBP4 and HDAC1 to silence E-cadherin expression. ( A ) The mRNA expression levels of E-cadherin , N-cadherin , Vimentin , and MMP2 after KTN1-AS1 overexpression and knockdown. ( B ) The regulatory effect of KTN1-AS1 on protein levels of E-cadherin, N-cadherin, Vimentin, and MMP2 was detected by western blot. Original western blots were presented in Supplementary Fig. D, with blots cut prior to hybridization with antibodies. ( C ) Inhibition of RBBP4 increased the expression level of E-cadherin and partially reversed the regulation effect of KTN1-AS1 on the expression level of E-cadherin in Kyse150 and Kyse170 cells. ( D ) Co-IP assay was performed to examine the RBBP4-HDAC1 interaction in the groups with KTN1-AS1 overexpression and inhibition in Kyse150 and Kyse170 cells. Original western blots were presented in Supplementary Fig. E, with blots cut prior to hybridization with antibodies. ( E ) RIP assay showed the interaction between KTN1-AS1 and HDAC1 in Kyse150 and Kyse170 cells. ( F ) After transfection of pcDNA3.1-NC or pcDNA3.1-KTN1-AS1 for 12–24 h in Kyse150 and Kyse170 cells, then cells with pcDNA3.1-KTN1-AS1 were treated with or without 300 nM Trichostatin A (TSA) for additional 48 h, the mRNA expression of E-cadherin was detected by qRT-PCR method. ( G ) ChIP-qPCR was performed to detect the enrichment of ac-H3 in the promoter region of E-cadherin after overexpression and knockdown of KTN1-AS1 in Kyse150 cells. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Hybridization, Inhibition, Co-Immunoprecipitation Assay, Transfection, Quantitative RT-PCR, ChIP-qPCR

Journal: Scientific Reports

Article Title: KTN1-AS1 , a SOX2-mediated lncRNA, activates epithelial–mesenchymal transition process in esophageal squamous cell carcinoma

doi: 10.1038/s41598-022-24743-z

Figure Lengend Snippet: RBBP4 partially reverses the biological function of KTN1-AS1 on esophageal squamous cell carcinoma (ESCC) cells. ( A , B ) MTS and clone formation assays were performed to analyze the cell proliferation ability after co-transfected with pcDNA3.1-KTN1-AS1 and si-RBBP4 in Kyse150 cells. ( C , D ) Wound healing and transwell invasion assays were conducted to explore the migration and invasion ability after co-transfected with pcDNA3.1-KTN1-AS1 and si-RBBP4 in Kyse150 cells. Error bars are shown as mean ± SD from three replicate experiments (n = 3) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: RNA was immunoprecipitated with antibodies against RBBP4 (ZenBioScience, Cat# 385565) and HDAC1 (Proteintech, Cat# 10197-1-AP).

Techniques: Transfection, Migration

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: Clinicopathological characteristics of COAD patients.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques:

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: Prognostic value of mRNA expression of ATP6V1C2 in COAD patients. (A) The difference in ATP6V1C2 expression level between COAD and adjacent non-cancerous tissues based on TCGA COAD dataset; (B) Kaplan–Meier survival curves of overall survival (OS) based on the ATP6V1C2-high and -low groups expression in TCGA-COAD dataset; (C) Survival curves of OS based on the ATP6V1C2-high and -low groups expression in GSE29523 dataset; (D) Survival curves of OS based on the ATP6V1C2-high and -low groups expression in GSE71187 dataset. COAD: colon adenocarcinoma; TCGA: The Cancer Genome Atlas. *** indicates p < 0.001, statistical analysis was performed using two-tailed Student’s t -tests.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques: Expressing, Two Tailed Test

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: Protein–protein network of ATP6V1C2. (A) Protein–protein network between ATP6V1C2 and top 20 co-expressed genes predicted from GeneMANIA database; (B) Protein–protein network between ATP6V1C2 and DEGs in COAD among these top 20 genes. DEG: differentially expressed gene; COAD: colon adenocarcinoma.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques:

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: The association of ATP6V1C2 expression with EMT process. (A) The difference in ATP6V1C2 expression level among patients with stage I, stage II, stage III, and stage IV; (B) The difference in EMT score between ATP6V1C2-high and -low groups. ns indicates p > 0.05. * indicates p < 0.05. *** indicates p < 0.001. EMT, epithelial–mesenchymal transition. FPKM: Fragments per kilobase of exon model per million mapped fragments; FC: fold change; EMT: epithelial–mesenchymal transition.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: The difference in CD8 T effector pathway activity between ATP6V1C2-high and -low groups. (A) CD8 T effector pathway activity; (B) CD8 expression; (C) CD4 expression; (D) CXCL10 expression; (E) CXCL9 expression; (F) IFNG expression. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively. FPKM: Fragments per kilobase of exon model per million mapped fragments.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques: Activity Assay, Expressing

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: DEGs between ATP6V1C2-high and -low group and the potential roles of identified DEGs. (A) GO and KEGG enrichment of DEGs; (B) Wnt signaling pathway enriched from identified DEGs; (C) The associations between Wnt hub genes and cellular processes/signaling pathways implicated in tumor progression. DEGs: differentially expressed genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; EMT: epithelial–mesenchymal transition.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques: Protein-Protein interactions

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: In vitro studies on ATP6V1C2 in COAD tumorigenesis. (A) The protein expression level of ATP6V1C2 in human colonic epithelial cells (HCoEpiC) and COAD cells; (B) The mRNA expression level of ATP6V1C2 in SW480/HCT116 cells transfected with siATP6V1C2 and siNC; (C) Proliferation curves assessed by CCK8 assay during 120 h for ATP6V1C2 knockdown cell model of SW480; (D) Proliferation curves assessed by CCK8 assay during 120 h for ATP6V1C2 knockdown cell model of HCT116; (E) The cloning ability for ATP6V1C2 knockdown cell models of SW480 and HCT116; The expression level of Wnt pathway-related and EMT-related genes in SW480 (F) and HCT116 cells (G) with siATP6V1C2. COAD: colon adenocarcinoma. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively. OD: optical density; NC: normal control.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques: In Vitro, Expressing, Transfection, CCK-8 Assay, Knockdown, Cloning, Control

Journal: Frontiers in Genetics

Article Title: High Expression of ATP6V1C2 Predicts Unfavorable Overall Survival in Patients With Colon Adenocarcinoma

doi: 10.3389/fgene.2022.930876

Figure Lengend Snippet: Schematic diagram of ATP6V1C2 expression on the prognosis of COAD patients. ATP6V1C2 overexpression might promote EMT cellular process by activating Wnt signaling pathway, resulting in cancer metastasis and unfavorable prognosis for COAD patients. COAD: colon adenocarcinoma; EMT: epithelial–mesenchymal transition.

Article Snippet: Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was incubated with primary ATP6V1C2 (catalog number: 16274-1-AP) or GAPDH (catalog number: 10494-1-AP) polyclonal antibody (Proteintech, Rosemont, IL, United States), followed by HRP-conjugated secondary antibody.

Techniques: Expressing, Over Expression