Journal: Molecular Cancer

Article Title: CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

doi: 10.1186/s12943-021-01481-w

Figure Lengend Snippet: circRTN4 stabilizes RAB11FIP1 by preventing its ubiquitination and degradation. A Heat-map showing 99 circRTN4-interacting proteins in PDAC. Biotin-labelled circRTN4, RTN4 mRNA, circGFP were used to pull down circRTN4-interacting proteins. Mass spectrometry analysis was performed to identify the interacting proteins. B CircRTN4 interacted with RAB11FIP1 in PDAC cells, as revealed by RIP assay. C Bioinformatics analysis by PRIdictor revealed the RAB11FIP1-binding site (The seed region of interaction was in red) on circRTN4. Mutating RAB11FIP1-binding site on circRTN4 (The mutated seed region of interaction was in green) inhibited circRTN4-RAB11FIP1 interaction in PANC-1 cells, as revealed by circRNA pulldown assay. D CircRTN4 knockdown inhibited RAB11FIP1 expression in mice xenograft. E CircRTN4 knockdown did not affect RAB11FIP1 mRNA level in PDAC cells. F CircRTN4 knockdown decreased the stability of RAB11FIP1 after inhibition of protein synthesis by cycloheximide in PANC-1 cells. G 3-Dimensional structure of the circRTN4-RAB11FIP1 interaction revealed that circRTN4 blocked the ubiquitination site Lys578 of RAB11FIP1. H Immunoprecipitation with anti-RAB11FIP1 antibody in PANC-1 cells after circRTN4 knockdown, followed by immunoblotting analysis with anti-ubiquitin or anti-RAB11FIP1 antibody. CircRTN4 knockdown increased ubiquitination of RAB11FIP1. I RAB11FIP1 expression were upregulated in PDAC primary tumors and was positively correlated with circRTN4 level. Data represent mean ± SD from at least three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Briefly, cells were incubated magnetic beads labelled with antibody against RAB11FIP1 (rabbit; 16,778–1-AP Proteintech) or TWF1 (mouse; sc-376,539 Santa Cruz Biotechnology, TX, USA) overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Binding Assay, Knockdown, Expressing, Inhibition, Immunoprecipitation, Western Blot

Journal: Molecular Cancer

Article Title: CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

doi: 10.1186/s12943-021-01481-w

Figure Lengend Snippet: circRTN4 stabilizes RAB11FIP1 to promote EMT in PDAC. A N-cadherin expression was inhibited after circRTN4 knockdown in PANC-1 cells. B CircRTN4 knockdown in mice subcutaneous tumors inhibited N-cadherin expression. C Expression of EMT markers Slug, Snai1, Twist and Zeb1 were reduced after circRTN4 knockdown in PDAC cells. D Expression of Slug, Snai1, Twist and Zeb1 in circRTN4-overexpressing HPDE cells were rescued after RAB11FIP1 knockdown. E Slug, Snai1, Twist and Zeb1 expression were upregulated in PDAC primary tumors and were positively correlated with circRTN4 level. Data represent mean ± SD from at least three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Briefly, cells were incubated magnetic beads labelled with antibody against RAB11FIP1 (rabbit; 16,778–1-AP Proteintech) or TWF1 (mouse; sc-376,539 Santa Cruz Biotechnology, TX, USA) overnight at 4 °C.

Techniques: Expressing, Knockdown

Journal: bioRxiv

Article Title: The endocytic recycling pathway is controlled by the ADP-ribosylated GTPase Rab14

doi: 10.1101/2022.11.26.517555

Figure Lengend Snippet: Schematic representation of MARylated Rab14 role during endosomal progression. GTP-loaded Rab14 binds the effector RUFY1 on early/sorting endosomes. Endocytic stimuli, such as transferrin bound to its cognate receptor, cause PARP12 translocation to early/sorting endosomes, where Rab14 MARylation occurs. The ADP-ribose likely induces Rab14 conformational changes suitable for binding to the downstream effectors FIP1c/RCP (FIP1C) and Rab11, thus allowing endosome progression towards the endocytic recycling compartment (ERC). Here, GDP-bound Rab14 is released from endosomes, while GTP-loaded Rab11/FIP1C complex allows recycling of endosomes to the plasma membrane. At the ERC, an ADP-ribosylhydrolase (ADPrH) may remove the ADP-ribose moiety, making Rab14 available for the following cycle.

Article Snippet: Commercially available antibodies: mouse monoclonal antibodies to Transferrin Receptor/CD71 (clone H68.4; #13-6890, Thermo Fisher Scientific; western blots (WB) 1:3,000; immunofluorescence (IF) 1:200), EEA1 (#610456, BD Transduction Laboratories; WB 1:1,000; IF 1:100), Golgin-97 (CDF4, #A-21270, Thermo Fisher Scientific; IF 1:100); rabbit polyclonal antibodies to Rab4 (#ab13252, Sigma-Aldrich; IF 1:100), Rab14 (#R0656; Sigma-Aldrich, WB 1:2,000, IF 1:100), Rab11 (#71-5300, Thermo Fisher Scientific; IF 1:50), Rab11-FIP1 (#HPA025960, Atlas Antibodies; WB 1:1,000, IF 1:100), RUFY1 (#PA5-31400, Thermo Fisher Scientific; IF 1:100), PARP12 (#HPA063872, Sigma-Aldrich Prestige Antibodies, WB 1:2000); goat polyclonal antibody to PARP12 (#ab45873, Abcam, IF 1:200); HRP-conjugated secondary antibodies (Calbiochem), HRP-conjugated anti-rabbit secondary antibody, heavy Chain Specific (Sigma-Aldrich); AlexaFluor-488,-568 and -647 conjugated secondary antibodies and AlexaFluor -488, -568, -633 or -647 Conjugate transferrins (Thermo Fisher Scientific, Invitrogen).

Techniques: Translocation Assay, Binding Assay, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: The endocytic recycling pathway is controlled by the ADP-ribosylated GTPase Rab14

doi: 10.1101/2022.11.26.517555

Figure Lengend Snippet: (a) In vitro ADP-ribosylation assay using GST-tagged purified PARP12 catalytic fragment and His-tagged purified Rab14 wild-type (Rab14 WT) or its MARylation defective mutant E159Q-E162Q (Rab14 MUT), in presence of 4 μCi of [ P]-NAD + and 30 μM of total NAD + . Reactions were stopped at different times (as indicated); the incorporated [ P]-ADP-ribose was detected by autoradiography (AR [ P]). Lower panels show total levels of Rab14 and PARP12. (b) Quantifications of in vitro MARylated Rab14 are reported in the graph. Data represent the mean ± S.D. (N = 3 independent experiments, two-sided one-sample t-test; *P <0,05). (c) Representative Af1521 macro domain pull-down assay of total lysates from HeLa cells transfected with EGFP-tagged Rab14 WT (WT) or its MARylation defective mutants (E159Q, E162Q, E159Q-E162Q). Af1521 bound proteins (Af1521 macro domain pull-down) and total cell lysates (inputs) were separated by SDS-PAGE and analyzed by western blotting with a Rab14 antibody. (d) Quantifications of MARylated Rab14 relative to (c) are reported in the graph. Data represent the mean ± S.D. (N = 3 independent experiments; one way Analysis of Variance; ***P <0,0001). (e) Representative confocal microscopy images of transferrin internalization (Alexa-568-Tfn) in HeLa cells transfected with EGFP-tagged Rab14 WT or its MARylation defective mutant E159Q-E162Q (Rab14 MUT). (f) Quantification of cells showing internalized transferrin into perinuclear recycling endosomes on the total transferrin fluorescence is reported in the graph. Data represent the mean ± S.D. (N = 3 independent experiments; two-sided one-sample t-test; *P <0,0025). (g) Representative confocal microscopy images of double-transferrin internalization in HeLa cells transfected with EGFP-tagged Rab14 WT or its MARylation defective mutant E159Q-E162Q (Rab14 MUT). (h) Representative confocal microscopy images of Alexa-633-Transferrin (Tfn) uptake in HeLa cells upon overexpression of EGFP-tagged Rab14 WT or its MARylation defective mutant E159Q-E162Q (Rab14 MUT). Cells were processed for immunofluorescence and Rab11 localization analyzed using a Rab11 antibody (red). Merged signals are also reported. Scale bars, 10 μM.

Article Snippet: Commercially available antibodies: mouse monoclonal antibodies to Transferrin Receptor/CD71 (clone H68.4; #13-6890, Thermo Fisher Scientific; western blots (WB) 1:3,000; immunofluorescence (IF) 1:200), EEA1 (#610456, BD Transduction Laboratories; WB 1:1,000; IF 1:100), Golgin-97 (CDF4, #A-21270, Thermo Fisher Scientific; IF 1:100); rabbit polyclonal antibodies to Rab4 (#ab13252, Sigma-Aldrich; IF 1:100), Rab14 (#R0656; Sigma-Aldrich, WB 1:2,000, IF 1:100), Rab11 (#71-5300, Thermo Fisher Scientific; IF 1:50), Rab11-FIP1 (#HPA025960, Atlas Antibodies; WB 1:1,000, IF 1:100), RUFY1 (#PA5-31400, Thermo Fisher Scientific; IF 1:100), PARP12 (#HPA063872, Sigma-Aldrich Prestige Antibodies, WB 1:2000); goat polyclonal antibody to PARP12 (#ab45873, Abcam, IF 1:200); HRP-conjugated secondary antibodies (Calbiochem), HRP-conjugated anti-rabbit secondary antibody, heavy Chain Specific (Sigma-Aldrich); AlexaFluor-488,-568 and -647 conjugated secondary antibodies and AlexaFluor -488, -568, -633 or -647 Conjugate transferrins (Thermo Fisher Scientific, Invitrogen).

Techniques: In Vitro, Purification, Mutagenesis, Autoradiography, Pull Down Assay, Transfection, SDS Page, Western Blot, Confocal Microscopy, Fluorescence, Over Expression, Immunofluorescence

Journal: Molecular Medicine

Article Title: The transcription factor Stat-1 is essential for Schwann cell differentiation, myelination and myelin sheath regeneration

doi: 10.1186/s10020-023-00667-w

Figure Lengend Snippet: Stat1 may recruit Rab11fip1 to regulate SC differentiation. A Stat1-binding regions obtained by ChIP-seq. The colored rectangle annotation represent peaks at the promoter (defined as ≤ 1000 bp), the promoter (defined as 1000 − 3000 bp), downstream of thepromoter (defined as ≤ 300 bp), 5’ UTR, 3’UTR, exon, intron or distal intergenic. B Distribution of enrichment intensity of ChIP-seq reads in the proximity of the transcription start site (TSS) (up), and consensus motifs at Stat1 bound sequences (down). C GO enrichment analysis of peak-related genes (bubble diagram). D Venn diagram shows overlap of genes with top 500 peak in the promoter related genes and 386 differentially expressed genes ( p < 0.05 and Fold change ≥ 2) between Stat1 knockdown groups and the control groups. E Histograms visualizing Stat1 binding around the 4 overlapping genes (Ano1, Nts, C1qb and Rab11fip1) locus. “Peak number” represents the plausibility ranking of the peak. F ChIP-qPCR for Stat1 occupancy on the Ano1, Nts, C1qb and Rab11fip1 promoters in differentiating SCs. T -test, ** p < 0.01, *** p < 0.01, n = 3 per group. G Luciferase activity of Ano1, Nts, C1qb and Rab11fip1 promoter and mutant promoter in HEK293 cells co-transfected with pGV141-Stat1 (Stat1 overexpression plasmid) plus an plasmid containing the Renilla luciferase gene. T -test, *** p < 0.01, n = 3 per group. H Western blots showing the expression level of Rab11fip1 and MAG in differentiated SCs treated with Rab11fip1-siRNA and Scramble. The histograms showing that knockdown Rab11fip1 reduces MAG expression in differentiated SCs. T -test, *** p < 0.001 vs Scramble, n = 3 per group

Article Snippet: For short interfering RNA (siRNA) transfection, approximately 1 × 10 cells SCs were seeded in 24-well culture plates and cultivated for 24 h. The siRNAs including Stat1-siRNAs or Rab11fip1-siRNAs, and their non-targeting negative control (RiboBio) were transfected into cells using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA).

Techniques: Binding Assay, ChIP-sequencing, Knockdown, Control, Luciferase, Activity Assay, Mutagenesis, Transfection, Over Expression, Plasmid Preparation, Western Blot, Expressing