Journal: Cancer Management and Research

Article Title: Long Noncoding RNA CCAT1 Functions as a Competing Endogenous RNA to Upregulate ITGA9 by Sponging MiR-296-3p in Melanoma

doi: 10.2147/CMAR.S252635

Figure Lengend Snippet: The expression level of CCAT1 in melanoma tissues and cells. ( A and B ) RT-qPCR assay was performed to examine the expression level of CCAT1 in 40 pairs of melanoma tissues and adjacent normal tissues, as well as in melanoma cell lines (A375, SK-MEL-28, and A875) and normal human epidermal melanocytes (NHEM cells). * P < 0.05.

Article Snippet: Melanoma cell lines (A375, SK-MEL-28, and A875) and normal human epidermal melanocytes (NHEM) were obtained from China center for type culture collection (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk

doi: 10.1038/s41598-019-46529-6

Figure Lengend Snippet: Heat Map of proteins regulated in keratinocytes after transfection with different miRNAs as revealed by iTRAQ mass spectrometry. Proteins have been grouped into different networks and their relative expressions at protein level are depicted where fold change of ≤0.8 is shown as downregulated in blue colour and fold change ≥1.2 is shown as upregulated in pink colour. ( A ) Oxidative stress Proteins; ( B ) Mitochondrial proteins; ( C ) Keratins & Structural proteins; ( D ) Apoptosis proteins; ( E ) Vesicular trafficking partners; ( F ) Miscellaneous protein including Immune System proteins & related Kinases.

Article Snippet: Normal Human epidermal melanocyte and keratinocyte cultures were kind gift from Dr. T. N. Vivek, Institute of Genomics and Integrative Biology, New Delhi, who had purchased them from M/s Lonza (India).

Techniques: Transfection, Multiplex sample analysis, Mass Spectrometry

Journal: Scientific Reports

Article Title: Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk

doi: 10.1038/s41598-019-46529-6

Figure Lengend Snippet: Expression of Tyrosinase related protein 1 (TRP1) in keratinocytes: ( a , b ) show relative expression of TRP1 protein in six miRNA transfected Keratinocytes compared with control as revealed by iTRAQ Mass Spectrometry. ( c ) Expression of TRP1 at protein level in Keratinocyte cell lysates in K1, K2 and K3 samples shows variable expression. M is Melanocyte lysate used as positive control where TRP1 expression is very abundant, N is NIH3T3, negative control, where there is no expression of TRP1.Full gel pictures of this cropped gel are shown in Supplementary Fig. . The lower Immunoblot represents β-Actin as loading control. ( d ) Expression of TRP2/DCT in NIH3T3 (N), melanocytes (M) and two samples of keratinocyte cultures K1 and K2, to show the expression of the melanogenic protein only in melanocytes and not in keratinocytes, full gel pictures are shown in Supplementary Fig. .

Article Snippet: Normal Human epidermal melanocyte and keratinocyte cultures were kind gift from Dr. T. N. Vivek, Institute of Genomics and Integrative Biology, New Delhi, who had purchased them from M/s Lonza (India).

Techniques: Expressing, Transfection, Control, Multiplex sample analysis, Mass Spectrometry, Positive Control, Negative Control, Western Blot

Journal: Scientific Reports

Article Title: Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk

doi: 10.1038/s41598-019-46529-6

Figure Lengend Snippet: Downregulation of Tyrosinase related protein 1 (TRP1) by selected miRNA at mRNA and protein levels. ( a ) TRP1 mRNA profiling was done by qPCR and normalized with NC (negative control i.e., scrambled control). Expression of TRP1 in keratinocytes at mRNA level after transfection with pre-miR-326, pre-miR-518a-5p and pre-miR-518c, ( b ) Transcriptional expression of TRP1 in keratinocytes after transfection with pre-miR-185, pre-miR-202-3p, pre-miR-423-3p and pre-miR-525-5p ( c ) Western blot of TRP1 expression after transfection with pre-miR-326, pre-miR-518a-5p, pre-miR-518c. UT is Untransfected control. Full gel pictures are shown in Supplementary Fig. . ( d ) Western blot of TRP1 expression after transfection with pre-miR-185, pre-miR-202-3p, pre-miR-423-3p and pre-miR-525-5p. Full gel pictures are shown in Supplementary Fig. . ( e ) Densitometric analysis of TRP1 protein expression in pre-miR-326, pre-miR-518a-5p, pre-miR-518c transfected keratinocytes after normalization with β- Actin, ( f ) Densitometric analysis of TRP1 protein expression in pre-miR-185, pre-miR-202-3p, pre-miR-423-3p and pre-miR-525-5p transfected keratinocytes after normalization with β-Actin (N = 3), ( g ) Target validation assay results showing significantly reduced relative luciferase activity of 3'UTR clones transfected with pre-miR-202-3p, pre-miR-518a-5p and pre-miR-525-5p. All the bars showing reduced expression are depicted as mean ± standard error of mean (S.E.M).

Article Snippet: Normal Human epidermal melanocyte and keratinocyte cultures were kind gift from Dr. T. N. Vivek, Institute of Genomics and Integrative Biology, New Delhi, who had purchased them from M/s Lonza (India).

Techniques: Negative Control, Control, Expressing, Transfection, Western Blot, Biomarker Discovery, Luciferase, Activity Assay, Clone Assay

Journal: Scientific Reports

Article Title: Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk

doi: 10.1038/s41598-019-46529-6

Figure Lengend Snippet: Melanosome transfer from melanocytes to keratinocytes after transfection of keratinocytes with selected pre-miRs. ( a ) Representative image for pellet color in pure Keratinocyte culture (NHEK, with no pigment) and keratinocytes transfected with miRNAs, co-cultured with melanocytes and differentially trypsinized to get only pure keratinocytes. Pellet color is lighter in transfected Keratinocytes co-cultured with Melanocytes as compared to scrambled control (NC). UT is Untransfected control. ( b ) Bar graph showing percentage decrease in Melanin content in keratinocytes that were transfected with, pre-miR-202, pre-miR-518a-5p and pre-miR-525-5p, co-cultured with melanocytes and obtained 48 hours post co-culture by differential trypsinization. As compared to the scrambled control i.e. NC (100%, 8.03 ± 3.1 µg), miR-202 showed 76.32 ± 7.6% (6.48 ± 3.1 µg), miR-518a showed 73.2 ± 8.4% (6.21 ± 2.98 µg) and miR-525 showed 67.75 ± 7.3% (5.89 ± 2.8 µg) melanin content ( c ) Bar graph showing absolute melanin content in the keratinocyte cultures mentioned in ( b ) All the bars showing reduced expression are depicted as mean ± S.E.M mentioned above.

Article Snippet: Normal Human epidermal melanocyte and keratinocyte cultures were kind gift from Dr. T. N. Vivek, Institute of Genomics and Integrative Biology, New Delhi, who had purchased them from M/s Lonza (India).

Techniques: Transfection, Cell Culture, Control, Co-Culture Assay, Expressing

Journal: Scientific Reports

Article Title: Micro RNAs upregulated in Vitiligo skin play an important role in its aetiopathogenesis by altering TRP1 expression and keratinocyte-melanocytes cross-talk

doi: 10.1038/s41598-019-46529-6

Figure Lengend Snippet: Melanosome transfer from melanocytes to TRP1 siRNA transfected keratinocytes. ( a ) Transcriptional expression of TRP1 in Normal human epidermal keratinocytes (NHEKs) transfected with TRP1 siRNA in 3 samples compared with scrambled control (NC). ( b ) Western blots for expression of TRP1 protein in the same 3 NHEK samples transfected with TRP1 siRNA (full gel pictures are shown in Supplementary Fig. . ( c ) Densitometric plots for TRP1 expression as compared with NC normalized with endogenous control β-Actin, confirm downregulation of TRP1 in siRNA transfected keratinocytes. ( d ) Representative image for pellet color in pure keratinocytes, TRP1 siRNA transfected keratinocytes that were co-cultured with melanocytes and harvested by differential trypsinization. Pellet color of keratinocytes transfected with siRNA and co-cultured with melanocytes, is lighter as compared to the keratinocytes transfected with scrambled control (NC) co-cultured with melanocytes. ( e , f ) Bar graphs showing percentage decrease in melanin and total melanin content in keratinocytes transfected with siRNA compared with that of keratinocytes transfected with scrambled control after co-culture with melanocytes. The melanin content in TRP1 siRNA transfected keratinocytes co-cultured with melanocytes was reduced from 100% (11.13 ± 2.87 µg) in NC to 71.68 ± 10.06% (8.53 ± 3.19 µg). While there is about 29% decrease, the percent decrease does not show statistically significant difference since for all the samples the reduction was calculated from 100%. However, absolute melanin content clearly shows significant reduction in melanin content in siRNA transfected keratinocytes.

Article Snippet: Normal Human epidermal melanocyte and keratinocyte cultures were kind gift from Dr. T. N. Vivek, Institute of Genomics and Integrative Biology, New Delhi, who had purchased them from M/s Lonza (India).

Techniques: Transfection, Expressing, Control, Western Blot, Cell Culture, Co-Culture Assay