human rab7 protein  (Cell Signaling Technology Inc)

Journal: Nature communications

Article Title: Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis.

doi: 10.1038/s41467-024-54324-9

Figure Lengend Snippet: Fig. 1 | Expression of Rab7a in melanoma versus non-melanoma cancer cells, correlation with TPC2 expression, and interaction of Rab7a with TPC2. a Gene expression profile (qPCR) of Rab7a in human melanoma lines compared to differ- ent non-melanoma cancer lines. Error bars represent mean values ± SEM. Data points represent biological replicates. b Protein levels corroborating mRNA expression data in melanoma lines (compared to the breast cancer line MDA-MB- 231). Shown are biological replicates, each. c Gene expression profile (qPCR) of Rab7b in human melanoma lines compared to different non-melanoma cancer lines. Data points show biological replicates. Error bars represent mean values ± SEM. Data points represent biological replicates. d Melanoma lines showing strong expression correlation between Rab7a and TPC2 but not TPC1. e Gene expression profile (qPCR) showing relative expression of TPCs in human melanoma lines

Article Snippet: The following antibodies were used for human Rab7 protein from Cell Signaling Technology: 2094S and 9367S, binding around residues Asp193 and Glu188, respectively, corresponding to Exon 6.

Techniques: Expressing, Gene Expression

Journal: Nature communications

Article Title: Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis.

doi: 10.1038/s41467-024-54324-9

Figure Lengend Snippet: Fig. 3 | Effect of Rab7 inhibitor on TPC2 activity and physical interaction of Rab7a with TPC2. a, b Inhibition of PI(3,5)P2 evoked currents in endolysosomes (EL), expressing hTPC2 alone or with Rab7a, using the Rab7-inhibitor CID1067700. Shown are representative current density-voltage relationships of enlarged EL, expressing hTPC2WT + hRab7WT or hTPC2WT alone, activated with 1 µM PI(3,5)P2 followed by application of CID1067700 (diff. conc.) and 1 mM ATP (max. effect). c Statistical summary of data as shown in (a, b) at −100 mV. Each dot represents a single current density value measured from one EL. Data were tested for statistical significance with one-way ANOVA test followed by Tukey’s post-test (***p < 0.001, ****p < 0.0001, n = 3). d Cartoon showing CRISPR/Cas9 strategy to knockout TPCN2 in the SK-MEL-5 cell line. e qPCR data showing relative expression of TPC2 in WT and TPC2 KO SK-MEL-5 (n = 3). f Statistical summary of data (average current densities at −100 mV) as shown in (g) (n = 6). g Representative current density-voltage relationships from −100 to +100 mV showing basal, TPC2-A1-P (20 µM) activated and ATP (1 mM) blocked currents.

Article Snippet: The following antibodies were used for human Rab7 protein from Cell Signaling Technology: 2094S and 9367S, binding around residues Asp193 and Glu188, respectively, corresponding to Exon 6.

Techniques: Activity Assay, Inhibition, Expressing, CRISPR, Knock-Out

Journal: Nature communications

Article Title: Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis.

doi: 10.1038/s41467-024-54324-9

Figure Lengend Snippet: Fig. 6 | Expression of MITF and GSK3β in different melanoma lines and effects of Rab7a or TPC2 KOor small molecule blockers. a Representative Western blots for MITF and Rab7 protein expression in different melanoma lines and in the breast cancer line MDA-MB-231, normalized to Vinculin. b, c Correlation plot for MITF/Rab7 expression (b) and statistical analysis of experiments as shown in (a) (mean values ± SEM, n = 5–8). Data points represent biological replicates (c). d Representative images of sections from healthy lymphnode (male, abdomen) and melanoma lymphnode metastasis (male, iliacal) samples stained with hMITF antibody (IHC). Scale bars = 5 mm. e IHC evaluation was carried out considering the percentage of stained tumor cells. Statistical significance was assessed by two- tailed unpaired t-test, *p = 0.0125 (mean ± SD). One dot corresponds to one independent human donor (n = 10 for each condition). Genetic knockout of either Rab7a (f, g) or TPC2 (h, i) in SK-MEL-5 cells shows reduction in the protein levels of

Article Snippet: The following antibodies were used for human Rab7 protein from Cell Signaling Technology: 2094S and 9367S, binding around residues Asp193 and Glu188, respectively, corresponding to Exon 6.

Techniques: Expressing, Western Blot, Staining, Two Tailed Test, Knock-Out

Journal: Nature Communications

Article Title: α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation

doi: 10.1038/s41467-021-21882-1

Figure Lengend Snippet: a Scatterplot of all proteins uniquely identified in both SILAC experiments (L light, M medium, H heavy amino acids). Fold change ( x -axis and y -axis for experiment A and B, respectively) are shown as log2. Scatterplot of the proteins upregulated ≥ 2 fold under bafilomycin A1 (BafA1) treatment is shown on the right. b Representative α-catenins (CTNNA3 and CTNNA1) immunoblotting in double ATG7/10 knockdown MCF10A cells. The experiment was repeated at least twice with similar results. c CTNNA3/GAPDH (left panel) and CTNNA1/GAPDH (right panel) densitometry in MCF10A cells exposed to either control or ATG7/ATG10 siRNAs. The graphs show the mean ± s.d. (CTNNA3/ GAPDH, n = 3 independent experiments) and mean ± s.e.m. (CTNNA1/ GAPDH, n = 5 independent experiments; *** P < 0.001, * P < 0.05; two-tailed one sample t -test). d Representative immunoblots of Ctnna3 levels in wild-type and Atg16L1 hypomorph (Atg16 hyp) primary cortical neurons. The graph shows the Ctnna3/Gapdh levels: mean ± s.d. ( n = 3 independent experiments; * P < 0.5; two-tailed one sample t -test). e Representative α-catenins (CTNNA3 and CTNNA1) immunoblots in MCF10A cells under starvation with EBSS (6 h). The experiment was repeated at least twice with similar results. f CTNNA3/GAPDH (left panel) and CTNNA1/GAPDH (right panel) densitometry in MCF10A cells starved in EBSS for 6 h. The graphs show the mean ± s.d. ( n = 3 independent experiments; ** P < 0.01, * P < 0.05; two-tailed one sample t -test). g Representative α-catenins (CTNNA3 and CTNNA1) immunoblots in MCF10A cells treated with the autophagy inducer Tat-Beclin1 peptide (20 µM for 48 h). Tat-scrambled peptide was used as control. The experiment was repeated at least twice with similar results. h Representative immunoblot of mEm-CTNNA1 in MCF10A cells exposed to BafA1 (200 nM, 16 h). The MCF10A cells were transiently transfected with mEm-CTNNA1. The graph shows the mEm-CTNNA1/GAPDH levels: mean ± s.d. ( n = 6 independent experiments; * P < 0.05; two-tailed one sample t -test). i Representative immunoblot of mEm-CTNNA1 in MCF10A cells exposed to autophagy inducers: SMER28 (20 µM) and EBSS for 6 h. The graph shows the mEm-CTNNA1/GAPDH levels: mean ± s.d. ( n = 3 independent experiments; ** P < 0.01, * P < 0.05; two-tailed one sample t -test). Exact P values for asterisks: c (from left to right) 0.0280, 0.0009; d 0.0461; f (from left to right) 0.0069, 0.0181; h 0.0301; and i (from left to right) 0.0340, 0.0036.

Article Snippet: Binding of LC3B (purified from E. coli ) to CTNNA1 (TP301776, Origene) was performed by incubation of recombinant proteins at 25 °C for 1 h, followed by immunoprecipitation with rabbit anti-CTNNA1 antibody (ab51032, Abcam, 1:100) and processed for WB analysis.

Techniques: Western Blot, Two Tailed Test, Transfection

Journal: Nature Communications

Article Title: α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation

doi: 10.1038/s41467-021-21882-1

Figure Lengend Snippet: a Representative images of endogenous CTNNA3–LC3 colocalization in MCF10A cells treated with BafA1. The control cells were treated with DMSO. Scale bars are 10 µm. The graphs on the right show the image intensities for all channels (CTNNA3, LC3 and DAPI). Scale bars are 10 µm. The experiment was repeated twice with similar results. b Quantification of colocalization for the experiment in ( a ). The Pearson’s and Mander’s (fraction of CTNNA3 overlapping LC3) coefficients were quantified for n = 45 cells (DMSO) and n = 38 cells (BafA1). Bars represent the mean ± s.e.m. (**** P < 0.0001; two-tailed t -test). c Representative confocal images of endogenous CTNNA3–LC3 colocalisation in MCF10A cells exposed to either Tat-scrambled or Tat-beclin1 peptide (20 µM, 48 h). Scale bars are 10 µm. The graph on the right shows the fluorescent intensity for each channel (red-CTNNA3, green-LC3 and blue-DAPI) along the indicated distances. d Co-immunoprecipitation of endogenous LC3 with CTNNA1 in BafA1-treated MCF10A cells. The experiment was repeated three times with similar results. e Co-immunoprecipitation of endogenous LC3 with wild-type and Flag-CTNNA1 mutants in BafA1-treated MCF10A cells. MCF10A cells were initially transfected with empty Flag (control vector), wild-type Flag-CTNNA1 or the indicated Flag-CTNNA1 mutants. The Flag-tagged proteins were pulled down using the Flag-Trap technology. The experiment was repeated three times with similar results. f Co-immunoprecipitation of endogenous LC3 with wild-type and mEm-CTNNA1 mutants. MCF10A cells were transfected with empty mEm (control vector), wild-type mEm-CTNNA1 or the indicated mEm-CTNNA1 mutants. The mEm-tagged proteins were pulled down using the GFP-Trap technology. The experiment was repeated three times with similar results. g 3D structure of the LIR region (417 KE Y AQ V 422) and its conservation among various species of α-catenins. h Representative images of mEm-CTNNA1—endogenous LC3 colocalization in MCF10A cells transiently transfected with either wild-type or Y419A-V422A mEm-CTNNA1. Scale bars are 10 µm. i The Pearson’s (PC), Overlap (OC) and Mander’s (M1—fraction of LC3 overlapping mEm-CTNNA1, and M2—fraction of mEm-CTNNA1 overlapping LC3) coefficients for MCF10A cells treated as in ( h ). Thirty-one wild-type cells and 21 Y419A-V422A mutant cells were quantified over three independent experiments. Bars represent the mean ± s.e.m. (**** P < 0.0001, *** P < 0.001; two-tailed t -test). Exact P values for asterisks: i 0.0009.

Article Snippet: Binding of LC3B (purified from E. coli ) to CTNNA1 (TP301776, Origene) was performed by incubation of recombinant proteins at 25 °C for 1 h, followed by immunoprecipitation with rabbit anti-CTNNA1 antibody (ab51032, Abcam, 1:100) and processed for WB analysis.

Techniques: Two Tailed Test, Immunoprecipitation, Transfection, Plasmid Preparation, Mutagenesis

Journal: Nature Communications

Article Title: α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation

doi: 10.1038/s41467-021-21882-1

Figure Lengend Snippet: a Representative immunoblots of MCF10A cells transiently expressing wild type, L897A-F900A, Y419A-V422A or Y419A-V422A-L897A-F900A mEm-CTNNA1 constructs and exposed to BafA1 treatment (400 nM, 6 h). GFP/GAPDH densitometry is shown on the right: mean ± s.d. ( n = 5 independent experiments; * P < 0.05, ns not significant; two-tailed one sample t -test). b Representative immunoblot of MCF10A cells expressing Flag-wild-type and the indicated CTNNA1 mutants upon BafA1 treatment (400 nM, 6 h). c Representative immunoblots of MCF10A cells transiently expressing wild-type or Y419A-V422A mEm-CTNNNA1 and exposed to autophagy inducers: SMER28 (20 µM) and EBSS for 6 h. The graphs show the GFP/GAPDH densitometry of one representative experiment: mean ± s.d. ( n = 3 independent experiments; ** P < 0.01, * P < 0.05, ns not significant; two-tailed one sample t -test). The experiment was repeated with similar results. d Representative immunoblot of MCF10A cells expressing Flag-wild-type and the indicated CTNNA1 mutants upon exposure to different concentrations of SMER28 (20 and 50 µM) for 24 h. e Representative immunoblot of MCF10A cells expressing Flag-wild-type and the indicated CTNNA1 mutants upon exposure to EBSS for 4 h. Bars represent the mean ± s.e.m. ( n = 4 independent experiments; * P < 0.05, ns not significant; two-tailed one sample t -test). Exact P values for asterisks: a (from left to right) 0.0317, 0.1743, 0.4750, 0.3386; c (from left to right) 0.0496, 0.0096, 0.5166, 0.1364; e (from left to right) 0.0336, 0.9923, 0.3824, 0.4799.

Article Snippet: Binding of LC3B (purified from E. coli ) to CTNNA1 (TP301776, Origene) was performed by incubation of recombinant proteins at 25 °C for 1 h, followed by immunoprecipitation with rabbit anti-CTNNA1 antibody (ab51032, Abcam, 1:100) and processed for WB analysis.

Techniques: Western Blot, Expressing, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation

doi: 10.1038/s41467-021-21882-1

Figure Lengend Snippet: a Representative confocal images of YAP/TAZ endogenous immunostaining in MCF10A cells expressing either wild-type or mutant (Y419A-V422A, L897A-F900A or Y419A-V422A-L897A-F900A) mEm-CTNNA1. Scale bars are 10 µm. The experiment was repeated three times with similar results. b YAP/TAZ localisation in MCF10A cells transfected with wild-type or mEm-CTNNA1 mutants. Bars represent the mean ± s.e.m. ( n = 4 independent experiments; *** P < 0.001; two-way ANOVA). c YAP/TAZ localisation in MCF10A cells transfected with wild-type or mutants of mEm-CTNNA1 upon BafA1 exposure (200 nM, 16 h). Around 100 cells were analysed per condition —* P < 0.05, ns not significant; chi-squared test. d YAP/TAZ localisation in MCF10A cells transfected with wild-type or mutant forms of mEm-CTNNA1 upon α-catenin knockdown. Bars represent the mean ± s.d. ( n = 3 independent experiments; **** P < 0.0001; two-way ANOVA). e TEAD luciferase activity in MCF10A cells expressing either wild-type or mutant (Y419A-V422A) Flag-CTNNA1. Bars represent the mean ± s.d. ( n = 3 independent experiments; ** P < 0.01, * P < 0.05; two-tailed one sample t -test). f Representative confocal images of BrdU-positive MCF10A cells transfected with wild-type or mutant forms of mEm-CTNNA1. Scale bars are 10 µm. The experiment was repeated three times with similar results. g Percentages of BrdU-positive cells in MCF10A cells transfected with wild-type or mutant forms of mEm-CTNNA1. Bars represent the mean ± s.e.m. ( n = 4 independent experiments; *** P < 0.001, ** P < 0.01, * P < 0.05; two-way ANOVA). h Size of MCF10A cells expressing either wild-type or the indicated mEm-CTNNA1 mutants. n = 25 cells per condition were analysed for measuring cell area using ZEN software. Bars represent the mean ± s.e.m (** P < 0.01, * P < 0.05; two-tailed t -test). Exact P values for asterisks: b 0.0003; e (from left to right) 0.0146, 0.0022, 0.0049; g (from left to right) 0.0043, 0.0207, 0.0019; h (from left to right) 0.0153, 0.0102, 0.0010.

Article Snippet: Binding of LC3B (purified from E. coli ) to CTNNA1 (TP301776, Origene) was performed by incubation of recombinant proteins at 25 °C for 1 h, followed by immunoprecipitation with rabbit anti-CTNNA1 antibody (ab51032, Abcam, 1:100) and processed for WB analysis.

Techniques: Immunostaining, Expressing, Mutagenesis, Transfection, Luciferase, Activity Assay, Two Tailed Test, Software

Journal: Nature Communications

Article Title: α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation

doi: 10.1038/s41467-021-21882-1

Figure Lengend Snippet: a Representative confocal images of endogenous YAP/TAZ and F-actin immunostaining in MCF10A cells. MCF10A cells were initially exposed to ATG7/10 siRNAs for 48 h, and only after, followed by α-catenin depletion using CTNNA1/CTNNA3 siRNAs (together with ATG7/10 siRNAs) for other 48 h. Scale bars are 10 µm. The experiment was repeated with similar results. b YAP/TAZ localisation in MCF10A cells treated as in ( a ). CTNNA1/3 KD partially rescues the YAP/TAZ localisation phenotype in ATG7/10 KD cells. The experiment was repeated with similar results ( n (from left to right) = 280/304/299/333 cells; ** P < 0.01, **** P < 0.0001; chi-square test). c Quantification of percentages of MCF10A cells with F-actin stress fibres. MCF10A cells were initially exposed to ATG7/10 siRNAs for 48 h, followed by α-catenin depletion using CTNNA1/CTNNA3 siRNAs for other 48 h. The experiment was repeated with similar results ( n (from left to right) = 202/ 200/ 206/ 233 cells; * P < 0.05, **** P < 0.0001; chi-square test). d Size of MCF10A cells initially exposed to ATG7/10 siRNAs for 48 h, followed by α-catenin depletion using CTNNA1/CTNNA3 siRNAs for other 48 h. Confocal images of 25 cells per condition were analysed for measuring cell area using ZEN software. Bars represent the mean ± s.e.m (**** P < 0.0001, ** P < 0.01; two-tailed t -test). e Mathematical modelling of YAP/TAZ activity when reducing autophagy levels. The graph shows the dependency of YAP/TAZ activity on the initial levels of α-catenins and the strength of the feedback loop involving YAP/TAZ control of autophagy v Y . f Representative immunoblot of α-catenin in various cell lines. Bars represent the mean ± s.d. ( n = 3 independent experiments; *** P < 0.001, ** P < 0.01, * P < 0.05; two-tailed one sample t -test). g Model for the autophagy-dependent YAP/TAZ activity in two initial conditions: low and high α-catenin levels. Autophagy inhibition in cells with high initial α-catenin levels promotes α-catenin accumulation which sequester YAP/TAZ into the cytosol (Hippo signalling is ON). However, autophagy inhibition in cells with low initial α-catenin levels causes only a small increase in these proteins, increase unable to sequester YAP/TAZ into the cytosol (as their levels also increase, being also autophagy substrates)—Hippo signalling is OFF. The feedback loop of YAP/TAZ controlling autophagy accentuates these effects. Exact P values for asterisks: b 0.0023; c 0.0105; d 0.0082; f (from left to right) <0.0001, 0.0027, <0.0001, 0.0027, 0.0276.

Article Snippet: Binding of LC3B (purified from E. coli ) to CTNNA1 (TP301776, Origene) was performed by incubation of recombinant proteins at 25 °C for 1 h, followed by immunoprecipitation with rabbit anti-CTNNA1 antibody (ab51032, Abcam, 1:100) and processed for WB analysis.

Techniques: Immunostaining, Software, Two Tailed Test, Activity Assay, Western Blot, Inhibition

Journal: Nature Communications

Article Title: α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation

doi: 10.1038/s41467-021-21882-1

Figure Lengend Snippet: a Representative immunoblots of CTNNA1 in multiple cell lines: MCF10A, HEK293T, HeLa, HepG2 after exposure to ATG7/10 siRNAs. Bars represent the mean ± s.e.m. ( n (from left to right) = 10/4/4/6 independent experiments; *** P < 0.001, * P < 0.05; two-tailed one sample t -test). b TEAD luciferase activity in MCF10A cells exposed to CTNNA1/3 siRNAs, followed by treatment with EBSS (6 h), as indicated. Bars represent the mean ± s.e.m. ( n = 6 independent experiments; *** P < 0.001, * P < 0.05; two-tailed one sample t -test). c TEAD luciferase activity in MCF10A cells exposed to CTNNA1/3 siRNAs, followed by treatment with Trehalose (100 mM, 24 h), as indicated. Bars represent the mean ± s.e.m. ( n = 9 independent experiments; *** P < 0.001, ** P < 0.01, * P < 0.05; two-tailed one sample t -test). d TEAD luciferase activity in MCF10A cells initially exposed to CTNNA1/3 siRNAs for 48 h followed by double ATG7/10 KD for other 48 h, as indicated. Bars represent the mean ± s.e.m. ( n = 6 independent experiments; ** P < 0.01; two-tailed one sample t -test). e TEAD luciferase activity in HepG2 cells transfected with mEm-empty or wild-type mEm-CTNNA1, and after, exposed to EBSS for 6 h. Bars represent the mean ± s.e.m. ( n = 4 independent experiments; ** P < 0.01, * P < 0.05, ns not significant; two-tailed one sample t -test). Exact P values for asterisks: a (from left to right) 0.0017, 0.0281, 0.0459, 0.0405; b 0.0001, <0.0001, 0.0160; c 0.0056, 0.0012, 0.0332; d 0.0046; e (from left to right) 0.0065, 0.0012, 0.0112.

Article Snippet: Binding of LC3B (purified from E. coli ) to CTNNA1 (TP301776, Origene) was performed by incubation of recombinant proteins at 25 °C for 1 h, followed by immunoprecipitation with rabbit anti-CTNNA1 antibody (ab51032, Abcam, 1:100) and processed for WB analysis.

Techniques: Western Blot, Two Tailed Test, Luciferase, Activity Assay, Transfection