Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: RAB11-UNC13D-FAK axis in recycling endosomes regulates the phosphorylation of FAK. A UNC13D knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by p-FAK, p-PXN, FAK, and PXN. Data are presented as the mean ± SD. The experiments were independently performed at least three times. Statistical analysis was performed using Student’s unpaired T test. ** p < 0.01, *** p < 0.001. B Co-immunoprecipitation analysis of the binding between exogenous Flag-UNC13D and HA-FAK in co-transfected HEK293 cells indicates the direct interaction of them. IgG was incubated with cell lysates as negative controls. C Immunoprecipitation of endogenous FAK from CAPAN-1 and PANC-1 cells confirms the binding between endogenous UNC13D and FAK. D Immunoprecipitation of endogenous RAB11 from PANC-1 overexpressing Flag-UNC13D cells indicates the binding between UNC13D and endogenous RAB11. E Co-immunoprecipitation analysis of the binding among RAB11, UNC13D, and FAK indicates the formation of RAB11-UNC13D-FAK complex. F UNC13D knockdown decreased the binding of RAB11 with FAK. RAB11 was immunoprecipitated from PANC-1 expressing shUNC13D or shSCR to examine the dependency of the interaction between RAB11 and FAK on UNC13D. G Co-localization of FAK, UNC13D, ITGB1, and RAB11 in PANC-1 cells. GFP-FAK, mOrange-UNC13D, and mCerulean-Rab11A cells were transduced. For the active integrin staining, 12G10 anti-ITGB1 was used. The data shown are representative of three independent experiments. Scale bar: 10 μm. H RAB11A knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by pFAK, pPaxillin (p-PXN), FAK, and Paxillin (PXN). The quantification of western blotting in ( G ) is presented as the mean ± SD of three independent experiments. Student’s unpaired T test, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Human PC cell lines (PANC-1, MIA PaCa-2, AsPC-1, CAPAN-1, and SNU213) and human embryonic kidney 293 (HEK293) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Immunoprecipitation, Binding Assay, Transfection, Incubation, Expressing, Staining, Western Blot

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: Analysis of the interaction domains of FAK and UNC13D. A Co-immunoprecipitation assay after the transduction of full-length FAK or its truncation mutants in HEK293 cells. Each group was purified by immunoprecipitation with HA beads, followed by a western blot to detect FLAG-UNC13D with the anti-FLAG antibody. B Co-immunoprecipitation analysis after transduction of full-length UNC13D or its truncation mutants in HEK293 cells. Each group was purified by immunoprecipitation with HA beads, followed by a western blot to detect FLAG-UNC13D with the anti-FLAG antibody

Article Snippet: Human PC cell lines (PANC-1, MIA PaCa-2, AsPC-1, CAPAN-1, and SNU213) and human embryonic kidney 293 (HEK293) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Co-Immunoprecipitation Assay, Transduction, Purification, Immunoprecipitation, Western Blot

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: The interaction of UNC13D with FAK is calcium-dependent. A The modulation of calcium-regulated FAK/PXN signaling was measured by the expression level of p-FAK, p-SRC, p-PXN, and p-ERK. The level of calcium was modulated by the treatment with ionomycin 1.25 μM or EGTA 1 mM for 5 min. B The interaction of UNC13D with FAK is modulated by the calcium. The binding between UNC13D and FAK in HEK293 cells was examined by immunoprecipitation after the modulation of calcium concentration by ionomycin 1.25 μM or EGTA 1 mM for 5 min. The quantification data were shown as mean ± SD of three independent experiments. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test. * p value < 0.001 ( C ) Mutations of calcium-binding domains (C2A or C2B) in UNC13D regulated its interaction with FAK. UNC13D wild type or C2 mutants (C2A or C2B) in HEK293 cells were immunoprecipitated with HA beads and immunoblotted by anti-FLAG antibody for the detection of Flag-UNC13D. The quantification data were shown as mean ± SD of three independent experiments. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparison test. ** p value < 0.001 ( D ) The abstract figure indicating recycling machinery of integrin coupled with FA turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells. Continuous recycling of integrin via exocytosis and endocytosis of vesicles is required for migration of cancer cells. Through exocytosis of endosomal vesicles, endocytosed integrin reaches at the plasma membrane of leading edge. During the assembly of FA, integrins interact with the extracellular matrix and recruit many proteins including talin, vinculin, paxillin and FAK. During the disassembly of FA, FA complex proteins are disintegrated and integrin is endocytosed into endosomal vesicles in a FAK-dependent manner. During the recycling process of integrin, UNC13D plays roles in tethering and priming of endosomal vesicles to the plasma membrane. Moreover, it can regulate disassembly of FA via RAB11-UNC13D-FAK axis. UNC13D knockdown inhibits exocytosis of endosomal vesicles and disassembly of FA, and finally cellular migration

Article Snippet: Human PC cell lines (PANC-1, MIA PaCa-2, AsPC-1, CAPAN-1, and SNU213) and human embryonic kidney 293 (HEK293) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Binding Assay, Immunoprecipitation, Concentration Assay, Comparison, Migration, Membrane, Knockdown

Journal: Science Advances

Article Title: A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates

doi: 10.1126/sciadv.adp3000

Figure Lengend Snippet: ( A ) Structural model of Ub (salmon) in complex with a Ub-conjugating enzyme E2 (yellow) and a Ub ligase E3 (RING) (green). The distance between the N termini of E3 and Ub is mentioned. Placing AP-tag at the N terminus of Ub and BirA at the catalytic end of RING-E3 ligase brings AP and BirA in proximity to each other. ( B ) Schematic representation of the Ub-POD method. ( C ) RAD18 Ub-POD workflow (left). HEK-293 cells transfected for 16 to 24 hours with AP-Ub and HA-BirA-RAD18 WT or HA-BirA-RAD18 C64D were exposed to UV (10 mJ/cm 2 ) (right). Biotin was added at the time of transfection, and cells were allowed to recover 6 hours after UV irradiation. Untreated cells served as control. Lysates were subjected to SDS-PAGE and immunoblotting. Results are representative of two independent biological replicates. ( D ) Volcano plot of streptavidin pulldown enriched proteins isolated from cells transfected with HA-BirA-RAD18 WT and AP-Ub in the absence or presence of UV exposure ( n = 3 biological replicates). Significantly altered proteins are shown in dark red or blue [false discovery rate (FDR) <0.05, log 2 FC >I0.6I] and light red or blue (FDR <0.05, 0 > log 2 FC < I0.6I) (moderated t test).

Article Snippet: Human embryonic kidney epithelial cell line 293 (HEK-293) (American Type Culture Collection CRL-1573) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v / v) heat-inactivated fetal bovine serum (Gibco) within a humidified 5% CO 2 incubator at 37°C.

Techniques: Transfection, Irradiation, Control, SDS Page, Western Blot, Isolation

Journal: Science Advances

Article Title: A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates

doi: 10.1126/sciadv.adp3000

Figure Lengend Snippet: ( A ) Left: Sequences of various AP-tag variants. Right: AP-tag variants were expressed in HEK-293 cells along with HA-BirA or HA-BirA-RAD18. Biotin was added at the time of transfection, and cells were allowed to recover 6 hours after UV irradiation. Lysates were subjected to SDS-PAGE and immunoblotting. Results are representative of three independent biological replicates. ( B ) HEK-293 cells were transfected with (−2)AP-Ub and HA-BirA or HA-BirA-RAD18 followed by UV exposure (10 mJ/cm 2 ). Cells were treated the same as in (A). Volcano plot of streptavidin enriched proteins in the two abovementioned conditions ( n = 3 biological replicates). Significantly altered proteins are shown in dark red or blue (FDR <0.05, log 2 FC >I0.6I) and light red or blue (FDR <0.05, 0 > log 2 FC < I0.6I) (moderated t test). ( C ) GO term analysis of potential RAD18 ubiquitination substrates. Bar graph shows significantly enriched pathways. ( D ) HEK-293 are transfected with indicated plasmids. Cells were treated, and lysates were immunoblotted the same as in (A). Results are representative of two independent biological replicates. ( E ) HEK-293 cells were transfected with indicated small interfering RNAs (siRNAs). After 48 hours, cells were exposed to UV (10 mJ/cm 2 ). The UV-treated and untreated cells were allowed to recover for 6 hours. Lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. Results are representative of three independent biological replicates. ( F ) Confocal microscopy of HEK-293 cells expressing either HA-BirA or HA-BirA-RAD18, along with (−2)AP-Ub, immunostained with anti–phospho histone H2AX (Ser 139 ) (green), antistreptavidin (red). Cells with notable Streptavidin signal are circled using white dotted lines. Scale bar, 12 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( G ) Percentage of transfected cells showing colocalization (Pearson’s coefficient > 0.5) of streptavidin and phospho histone H2AX (Ser 130 ) signals (indicated by yellow arrows) is represented (**** P < 0.0001) ( n , biological replicates = 2, 10 different fields).

Article Snippet: Human embryonic kidney epithelial cell line 293 (HEK-293) (American Type Culture Collection CRL-1573) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v / v) heat-inactivated fetal bovine serum (Gibco) within a humidified 5% CO 2 incubator at 37°C.

Techniques: Transfection, Irradiation, SDS Page, Western Blot, Confocal Microscopy, Expressing

Journal: Science Advances

Article Title: A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates

doi: 10.1126/sciadv.adp3000

Figure Lengend Snippet: ( A ) Biotin time course to estimate appropriate biotin treatment duration. HEK-293 cells were transfected with (−2)AP-Ub together with HA-BirA or HA-BirA-TRAF6 for 24 hours. Biotin (100 μM) was either added together with transfection mixture for 24 hours or added 6 hours, 3 hours, 1 hour, or 15 min before harvest, respectively. Notably, 15-min treatment was sufficient to induce strong biotinylation in HA-BirA-TRAF6 overexpressing cells and was therefore kept as treatment condition throughout all experiments. ( B ) Experimental setup for Ub-POD-TRAF6-MS. HEK-293 cells transiently overexpressing (−2)AP-Ub together with HA-BirA or HA-BirA-TRAF6 for 24 hours were subjected to biotin (100 μM) and PR-619 (10 μM) for 15 min and harvested. An aliquot was reserved for analysis via immunoblotting, the residual lysate was prepared for streptavidin pulldown and subjected to MS ( n = 4 biological replicates). ( C ) Volcano plot depicting altered biotinylated proteins after enrichment via streptavidin pulldown from HA-BirA-TRAF6 or HA-BirA expressing HEK-293 cells. Known and selected potential TRAF6 substrates are labeled. Validated hits depicted in 3E are highlighted in red. Significantly altered proteins are shown in dark red or blue (FDR < 0.05, log 2 FC > I1I) and light red or blue (FDR < 0.05, 0 > Ilog 2 FCI < I1I) (moderated t test, n = 4 independent experiments); ( D ) GO terms of proteins found to be significantly enriched in HA-BirA-TRAF6 versus HA-BirA with FC ≥1. Dot size correlates to number of proteins, dot color to term enrichment (FDR value). ( E ) Validation of TRAF6 substrates identified by Ub-POD-TRAF6-MS. HEK-293 cells transiently overexpressing (−2)AP-Ub together with HA-BirA, HA-BirA-TRAF6, or dimerization mutant HA-BirA-TRAF6F118A for 24 hours were subjected to biotin (100 μM) and PR-619 (10 μM) for 15 min. Lysates were subjected to streptavidin pulldown followed by SDS-PAGE and immunoblotting.

Article Snippet: Human embryonic kidney epithelial cell line 293 (HEK-293) (American Type Culture Collection CRL-1573) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v / v) heat-inactivated fetal bovine serum (Gibco) within a humidified 5% CO 2 incubator at 37°C.

Techniques: Transfection, Western Blot, Expressing, Labeling, Mutagenesis, SDS Page

Journal: Science Advances

Article Title: A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates

doi: 10.1126/sciadv.adp3000

Figure Lengend Snippet: ( A ) HEK-293 cells transfected for 16 to 24 hours with (−2)AP-Ub and indicated BirA-tagged constructs. Cells were treated with MG132 (10 μM) for 6 hours. Cells were kept in biotin (100 μM) the whole time. Lysates were separated by SDS-PAGE and analyzed by Western blotting. Results are representative of two independent biological replicates. ( B ) HEK-293 cells transfected for 24 hours with (−2)AP-Ub and BirA-HA or CHIP-GSGS-BirA-HA were incubated with biotin (100 μM) and MG132 (10 μM) for 6 hours. Lysates were subjected to streptavidin pulldown and MS analysis. Volcano plot of proteins labeled by CHIP-GSGS-BirA-HA and HA-BirA in 6 hours ( n = 3 biological replicates). Significantly altered proteins are shown in dark red or blue (FDR <0.05, log 2 FC > I0.6I) and light red or blue (FDR <0.05, 0 > log 2 FC < I0.6I) (moderated t test). ( C ) Bar graph representation of enriched GO terms for candidate CHIP substrates. ( D ) HA-BirA or CHIP-GSGS-BirA-HA transfected HEK-293 cells were cotransfected with either (−2)APUb or (−2)AP-UbΔGG. After 6 hours of MG132 treatment, whole-cell lysates were prepared followed by streptavidin pulldown. Pulldown and inputs were run on SDS-PAGE followed by immunoblotting with indicated antibodies. Results are representative of two independent biological replicates. ( E ) Knockdown of CHIP and immunoblotting for CHIP and ANXA5: HEK-293 cells were reverse transfected with either control siRNA (50 pmol) or different concentrations of CHIP siRNA (25 or 50 pmol). After 48 hours, cells were treated with either vehicle (dimethyl sulfoxide) or MG132 (10 μM). Whole-cell lysates, prepared after 6 hours of treatment, were run on SDS-PAGE followed by immunoblotting with the indicated antibodies. Results are representative of three independent biological replicates.

Article Snippet: Human embryonic kidney epithelial cell line 293 (HEK-293) (American Type Culture Collection CRL-1573) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v / v) heat-inactivated fetal bovine serum (Gibco) within a humidified 5% CO 2 incubator at 37°C.

Techniques: Transfection, Construct, SDS Page, Western Blot, Incubation, Labeling, Knockdown, Control

Journal: Science Advances

Article Title: A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates

doi: 10.1126/sciadv.adp3000

Figure Lengend Snippet: ( A ) BioID analysis of RAD18. HEK-293 cells transfected for 24 hours with BirA* or BirA*-RAD18 were exposed to UV (10 mJ/cm 2 ) and allowed to recover for 6 hours. Cells were kept in 100 μM biotin the whole time. Lysates were subjected to streptavidin pulldown followed by MS ( n = 3 biological replicates). Volcano plot of proteins labeled by BirA*-RAD18 and BirA*. Significantly altered proteins are shown in dark red or blue (FDR <0.05, log 2 FC > I1I) and light red or blue (FDR <0.05, 0 > log 2 FC < I1I) (moderated t test). ( B ) Bar graph depicting significantly enriched GO terms of hits from (A). ( C ) Overlap between hits identified in RAD18 BioID and Ub-POD experiments. ( D ) BioID analysis of CHIP. HEK-293 cells transfected for 24 hours with BirA* or CHIP-GSGS-BirA* were incubated with MG132 (10 μM) for 6 hours. Cells were kept in 100 μM biotin the whole time. Streptavidin pulldowns were performed with lysates followed by MS analysis ( n = 3 biological replicates). Volcano plot of proteins labeled by CHIP-GSGS-BirA* and BirA*. Significantly altered proteins are shown in dark red or blue (FDR <0.05, log 2 FC >I1I) and light red or blue (FDR <0.05, 0 > log 2 FC < I1I) (moderated t test). ( E ) Bar graph representation of significantly enriched GO terms with hits identified in (D). ( F ) Overlap between hits identified in CHIP BioID and Ub-POD experiments.

Article Snippet: Human embryonic kidney epithelial cell line 293 (HEK-293) (American Type Culture Collection CRL-1573) was cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v / v) heat-inactivated fetal bovine serum (Gibco) within a humidified 5% CO 2 incubator at 37°C.

Techniques: Transfection, Labeling, Incubation