Journal: bioRxiv

Article Title: Crosstalk in skin: Loss of desmoglein 1 in keratinocytes inhibits BRAF V600E -induced cellular senescence in human melanocytes

doi: 10.1101/2023.02.16.528886

Figure Lengend Snippet: A , Gene set enrichment analysis compared differentially expressed gene sets to published signatures of melanoma/melanocyte signaling. NES, normalized enrichment score. B , Volcano plots revealed log2 fold change against nominal p values (-log10) from differential expression analysis of mRNA sequencing of melanocytes with different BRAF statuses (control, WT BRAF and BRAF V600E ) and treated with control or Dsg1 knockdown conditioned media. Select genes involved in differentiation, pigmentation, and proliferation are marked.

Article Snippet: The following primary antibodies were used: BRAF (#9433), p44/42 MAPK (#9107), P-p44/42 MAPK (#4370), and p16 (#80772) from Cell Signaling Technology, BRAF V600E (ab200535) from Abcam, Dsg1 (#32-6000) from Thermo Fisher Scientific, GAPDH (G9545) from MilliporeSigma, and Tubulin (12G10) from Developmental Studies Hybridoma Bank.

Techniques: Quantitative Proteomics, Sequencing, Control, Knockdown

Journal: bioRxiv

Article Title: Crosstalk in skin: Loss of desmoglein 1 in keratinocytes inhibits BRAF V600E -induced cellular senescence in human melanocytes

doi: 10.1101/2023.02.16.528886

Figure Lengend Snippet: Human primary melanocytes were transduced with WT BRAF or BRAF V600E lentivirus, control or Dsg1-deficient CM were added 24 h after transduction and cells continued to be cultured in CM before they were harvested. A , Relative cell numbers over time. B , EdU flow cytometry assay to determine cell proliferation. The figures showed a clear separation of proliferating cells which have incorporated EdU in control and WT BRAF melanocytes but not in BRAF V600E melanocytes. C , Quantification of EdU flow cytometry assay. D , Annexin V/dead cell apoptosis flow cytometry assay was performed to detect cell death. E , Quantification of cell death by flow cytometry assay. F , Senescence-associated β-galactosidase activity was detected by flow cytometry assay. Right shift of the peak indicated the increase in senescence. G , Quantification of senescence flow cytometry assay. H , Expression of the indicated proteins was determined by Western blot. Results were reported as mean ± SD (n=3), statistical analysis was performed using two-way ANOVA with multiple comparisons (*, P < 0.05; ***, P < 0.001).

Article Snippet: The following primary antibodies were used: BRAF (#9433), p44/42 MAPK (#9107), P-p44/42 MAPK (#4370), and p16 (#80772) from Cell Signaling Technology, BRAF V600E (ab200535) from Abcam, Dsg1 (#32-6000) from Thermo Fisher Scientific, GAPDH (G9545) from MilliporeSigma, and Tubulin (12G10) from Developmental Studies Hybridoma Bank.

Techniques: Transduction, Control, Cell Culture, Flow Cytometry, Activity Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Crosstalk in skin: Loss of desmoglein 1 in keratinocytes inhibits BRAF V600E -induced cellular senescence in human melanocytes

doi: 10.1101/2023.02.16.528886

Figure Lengend Snippet: A , Bar graph of top enriched pathways represented in genes that were significantly up/down-regulated by Dsg1 knockdown CM. B , Heatmap with hierarchical clustering revealed the expression of the top 50 differentially expressed genes between BRAF V600E melanocytes treated with control and Dsg1-deficient CM.

Article Snippet: The following primary antibodies were used: BRAF (#9433), p44/42 MAPK (#9107), P-p44/42 MAPK (#4370), and p16 (#80772) from Cell Signaling Technology, BRAF V600E (ab200535) from Abcam, Dsg1 (#32-6000) from Thermo Fisher Scientific, GAPDH (G9545) from MilliporeSigma, and Tubulin (12G10) from Developmental Studies Hybridoma Bank.

Techniques: Knockdown, Expressing, Control

Journal: bioRxiv

Article Title: Crosstalk in skin: Loss of desmoglein 1 in keratinocytes inhibits BRAF V600E -induced cellular senescence in human melanocytes

doi: 10.1101/2023.02.16.528886

Figure Lengend Snippet: A , qRT-PCR was performed to confirm the up-regulation of NTN4 expression in BRAF V600E -transduced cells treated with Dsg1 knockdown CM. Mean ± SD depicted (n=3), statistical analysis was performed using Student’s t-test (**, p<0.01). B , Immunofluorescence analysis confirmed higher expression of Netrin-4 protein upon Dsg1-deficient CM treatment. C and D , qRT-PCR and immunofluorescence staining were carried out to confirm the knockdown of NTN4 expression (both mRNA and protein) by shRNAs. Results in C was reported as Mean ± SD (n=3), statistical analysis was performed using one-way ANOVA with multiple comparisons (***, p<0.001). E , Senescence-associated β-galactosidase expression was detected by X-gal staining assay. F , Quantification of X-gal staining assay. Mean ± SD depicted (n=3), statistical analysis was performed using two-way ANOVA with multiple comparisons (**, p<0.01).

Article Snippet: The following primary antibodies were used: BRAF (#9433), p44/42 MAPK (#9107), P-p44/42 MAPK (#4370), and p16 (#80772) from Cell Signaling Technology, BRAF V600E (ab200535) from Abcam, Dsg1 (#32-6000) from Thermo Fisher Scientific, GAPDH (G9545) from MilliporeSigma, and Tubulin (12G10) from Developmental Studies Hybridoma Bank.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Immunofluorescence, Staining