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: Molecular Oncology

Article Title: CHML is an NRF2 target gene that regulates mTOR function

doi: 10.1002/1878-0261.13194

Figure Lengend Snippet: Knockdown of CHML/ Rep2 decreases protein levels without affecting autophagy. (A) Immunoblot analysis of Rep2, p62, and LC3‐I/II protein levels in A549 cells treated with 5 n m of either NT or CHML /Rep2 siRNA for 72 h. (B) RFP‐GFP‐LC3 tandem fluorescence analysis of autophagy flux following siRNA knockdown same as (A). Yellow puncta = autophagosomes, red puncta = autolysosomes. Scale bar = 10 μm. (C) Immunoblot analysis of Rep2, p62, and LC3‐I/II protein levels in A549 cells transfected with 1 µg of either an empty vector (EV) or WT‐ CHML (Rep2 OE) for 24 h. (D) RFP‐GFP‐LC3 tandem fluorescent analysis of autophagy flux following Rep2 overexpression same as (C). Scale bar = 10 μm. (E) Immunoblot analysis of Rep2, LAMP1, Atg7, p62, Snap29, Rab7, and LC3‐I/II protein levels following Rep2 knockdown for 72 h. All groups for immunoblot and immunofluorescence analysis were performed in triplicate, and experiments were repeated two times to ensure validity of results.

Article Snippet: The primary antibodies against GAPDH (sc‐32233), NQO1 (sc‐32793), NRF2 (sc‐13032), RAB7 (sc‐376362) and phospho‐S6 (sc‐293144) were purchased from Santa Cruz.

Techniques: Knockdown, Western Blot, Fluorescence, Transfection, Plasmid Preparation, Over Expression, Immunofluorescence