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

Journal: Journal of Translational Medicine

Article Title: Papillary thyroid cancer organoids harboring BRAF V600E mutation reveal potentially beneficial effects of BRAF inhibitor-based combination therapies

doi: 10.1186/s12967-022-03848-z

Figure Lengend Snippet: List of drugs used in this study

Article Snippet: Primary antibodies against CK19 (1:200, Cat. No. Kit-0030, Maixin Biotech, China), galectin-3 (1:500, Cat. No. ab76245, Abcam), Ki-67 (1:200, cat. no. ab16667, Abcam), and BRAF (V600E mutant) (1:200, Cat. No. 29002, Cell Signaling Technology) were applied to the sections and incubated overnight at 4 °C.

Techniques: DNA Synthesis

Journal: Journal of Translational Medicine

Article Title: Papillary thyroid cancer organoids harboring BRAF V600E mutation reveal potentially beneficial effects of BRAF inhibitor-based combination therapies

doi: 10.1186/s12967-022-03848-z

Figure Lengend Snippet: Establishment of patient-derived PTC organoids harboring BRAF V600E mutation or wild-type (WT). a Overview of the procedure. Nine PTC organoids were derived and analyzed by histological characterization, DNA-sequencing, and drug sensitivity assays. b Expansion potential of nine PTC organoid cultures. Dots on the graph represent passage, arrows represent continuous expansion. PTC, papillary thyroid cancer; O, organoid. c Representative images of long-term cultured PTC organoids. Organoid cultures were derived from PTC-4_O and PTC-8_O. Scale bar, 100 µm. d Organoid formation efficiency of BRAF V600E PTC organoids and BRAF WT PTC organoids. Data represent the mean ± SEM of organoid number in PTC organoid lines. Scale bar, 100 µm

Article Snippet: Primary antibodies against CK19 (1:200, Cat. No. Kit-0030, Maixin Biotech, China), galectin-3 (1:500, Cat. No. ab76245, Abcam), Ki-67 (1:200, cat. no. ab16667, Abcam), and BRAF (V600E mutant) (1:200, Cat. No. 29002, Cell Signaling Technology) were applied to the sections and incubated overnight at 4 °C.

Techniques: Derivative Assay, Mutagenesis, DNA Sequencing, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Papillary thyroid cancer organoids harboring BRAF V600E mutation reveal potentially beneficial effects of BRAF inhibitor-based combination therapies

doi: 10.1186/s12967-022-03848-z

Figure Lengend Snippet: Immunofluorescence staining of CK19, galectin-3, BRAF V600E , and Ki-67 on PTC organoids and the parental tumors. Passage numbers of PTC organoid lines were: PTC-1_O, P3; PTC-2_O, P3; PTC-3_O, P2; PTC-4_O, P4; PTC-5_O, P3; PTC-6_O, P2; PTC-7_O, P4; PTC-8_O, P3; PTC-9_O, P4. PTC, papillary thyroid cancer. T, parental tumor; O, organoid. Scale bar, 100 µm

Article Snippet: Primary antibodies against CK19 (1:200, Cat. No. Kit-0030, Maixin Biotech, China), galectin-3 (1:500, Cat. No. ab76245, Abcam), Ki-67 (1:200, cat. no. ab16667, Abcam), and BRAF (V600E mutant) (1:200, Cat. No. 29002, Cell Signaling Technology) were applied to the sections and incubated overnight at 4 °C.

Techniques: Immunofluorescence, Staining

Journal: Journal of Translational Medicine

Article Title: Papillary thyroid cancer organoids harboring BRAF V600E mutation reveal potentially beneficial effects of BRAF inhibitor-based combination therapies

doi: 10.1186/s12967-022-03848-z

Figure Lengend Snippet: Histopathological Characteristics of PTC organoids with BRAF V600E mutation or wild-type and their parental tumors. Representative brightfield images of PTC organoids (top), and H&E staining of organoids (middle) and tumor tissues (bottom). Passage numbers of PTC organoid lines were: PTC-1_O, P3; PTC-2_O, P3; PTC-3_O, P2; PTC-4_O, P4; PTC-5_O, P3; PTC-6_O, P2; PTC-7_O, P4; PTC-8_O, P3; PTC-9_O, P4. PTC, papillary thyroid cancer; WT, wild-type. Scale bar, 100 µm

Article Snippet: Primary antibodies against CK19 (1:200, Cat. No. Kit-0030, Maixin Biotech, China), galectin-3 (1:500, Cat. No. ab76245, Abcam), Ki-67 (1:200, cat. no. ab16667, Abcam), and BRAF (V600E mutant) (1:200, Cat. No. 29002, Cell Signaling Technology) were applied to the sections and incubated overnight at 4 °C.

Techniques: Mutagenesis, Staining

Journal: Journal of Translational Medicine

Article Title: Papillary thyroid cancer organoids harboring BRAF V600E mutation reveal potentially beneficial effects of BRAF inhibitor-based combination therapies

doi: 10.1186/s12967-022-03848-z

Figure Lengend Snippet: Sensitivity of PTC-derived organoid lines for BRAF and MEK inhibitors. a Dose–response curves after 5 days of treatment with BRAF and MEK inhibitors. Each data point represents mean ± SEM of 3 independent biological replicates. IC 50 values are calculated and indicated beside the curve graphs. b Scatterplots of the correlation of 1-AUC values for targeted agents and chemotherapeutic drugs screened by two biological replicates (different passages of PTC organoids). Each data point represents 1-AUC for a PTC organoid line treated by the indicated drug. c Drugs with a common target have similar activity profiles across the PTC organoid lines. 1-AUC values are plotted for the inhibitors of BRAF V600E (vemurafenib and dabrafenib) and MEK (selumetinib and trametinib)

Article Snippet: Primary antibodies against CK19 (1:200, Cat. No. Kit-0030, Maixin Biotech, China), galectin-3 (1:500, Cat. No. ab76245, Abcam), Ki-67 (1:200, cat. no. ab16667, Abcam), and BRAF (V600E mutant) (1:200, Cat. No. 29002, Cell Signaling Technology) were applied to the sections and incubated overnight at 4 °C.

Techniques: Derivative Assay, Activity Assay