<p>Teneligliptin hydrobromide and retagliptin phosphate induce autophagy through TFEB translocation: (<b>A</b>,<b>B</b>) B16F1/GFP-LC3 cells were treated with either teneligliptin hydrobromide (TGN, 10, 100 μM), retagliptin phosphate (RGN, 10, 100 μM), or 3,4,5-trimethoxy cinnamate thymol ester (TCTE, 10 μg/mL). (<b>A</b>) Then, 24 h later, the treated cells were fixed to be imaged for green fluorescence. Cells in which autophagy was induced were analyzed by counting GFP-LC3 punctate dots under a confocal microscope. (<b>B</b>) The protein expression of LC3 was then examined by Western blotting. (<b>C</b>) B16F1 cells were treated with teneligliptin hydrobromide (TGN, 100 μM) or retagliptin phosphate (RGN, 100 μM), with or without bafilomycin A1 (Baf A1, 100 nM), for 6 h. The LC3 level was then examined by Western blotting. (<b>D</b>) B16F1/GFP-TFEB cells were exposed to teneligliptin hydrobromide (TGN, 100 μM) or retagliptin phosphate (RGN, 100 μM) for 24 h or Torin1 (0.25 μM) for 1 h. The cells were fixed for fluorescence imaging, and the nuclear translocalization of TFEB was assessed. The scale bar indicates 20 μm. (n = 3, ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001).</p> Full article ">Figure 2
<p>Teneligliptin hydrobromide and retagliptin phosphate induce anti-pigmentation in B16F1 cells treated with α-MSH: (<b>A</b>) B16F1 cells were treated with teneligliptin hydrobromide (TGN, 10, 100 μM) or retagliptin phosphate (RGN, 10, 100 μM) for 24 h, then cell viability was determined by CCK-8 assay. (<b>B</b>) B16F1 cells were pre-treated with alpha-melanocyte-stimulating hormone (α-MSH, 0.5 μM) for 36 h and then additionally exposed to teneligliptin hydrobromide (TGN, 10, 100 μM), retagliptin phosphate (RGN, 10, 100 μM) or 3,4,5-trimethoxy cinnamate thymol ester (TCTE, 10 μg/mL) for 24 h. (<b>B</b>) Melanin content is shown in cell pellets. (<b>C</b>) The melanin content was measured by assessing absorbance at 405 nm through a microplate reader, as outlined in the Materials and Methods Section. (n = 3, * <span class="html-italic">p</span> < 0.05 and *** <span class="html-italic">p</span> < 0.001).</p> Full article ">Figure 3
<p>Teneligliptin hydrobromide and retagliptin phosphate induce melanosomal degradation via autophagy: (<b>A</b>) First, 0.5 μM α-MSH-treated B16F1/TPC2-mRFP-EGFP cells were exposed to teneligliptin hydrobromide (TGN, 100 μM) or retagliptin phosphate (RGN, 100 μM) for 18 h, with or without 100nM bafilomycin (Baf A1) for 6 h. After fixation, the distribution of TPC2-mRFP-EGFP in the cells was imaged with confocal microscopy. (<b>B</b>) B16F1/TPC2-mRFP-EGFP cells were transfected with non-specific control siRNA (Sc) or siRNA targeting Atg5 (siAtg5). After 24 h transfection, the cells were further treated with 0.5 μM α-MSH for 36 h and then incubated with teneligliptin hydrobromide (TGN, 100 μM) or retagliptin phosphate (RGN, 100 μM). The number of RFP-only signals per cells was analyzed with merged images. The protein expression of Atg5 was then assessed by Western blotting. n = 3 and *** <span class="html-italic">p</span> < 0.001.</p> Full article ">Figure 4
<p>Inhibition of autophagy reverses the whitening effects of teneligliptin hydrobromide and retagliptin phosphate: (<b>A</b>,<b>B</b>) B16F1 cells were pre-treated with 0.5 μM α-MSH for 12 h and then the cells were incubated in the presence or absence of SBI-0206965 (SBI, 5 µM) for 24 h and additionally exposed to teneligliptin hydrobromide (TGN, 100 μM) or retagliptin phosphate (RGN, 100 μM) for 24 h. (<b>A</b>) Melanin content is shown in cell pellets. (<b>B</b>) Content in the cells was quantified by addressing absorbance at 405 nm using a microplate reader, as detailed in the Materials and Methods Section. (n = 3 and ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001).</p> Full article ">Figure 5
<p>Inhibition of autophagy decreases melanosomal degradation by teneligliptin hydrobromide and retagliptin phosphate: (<b>A</b>,<b>B</b>) B16F1 cells or B16F1/TPC2-mRFP-EGFP cells were pre-treated with 0.5 μM α-MSH for 12 h. And the cells were further incubated with or without SBI-0206965 (SBI, 5 µM) for 24 h and additionally exposed to teneligliptin hydrobromide (TGN, 100 μM) or retagliptin phosphate (RGN, 100 μM) for an additional 24 h. (<b>A</b>) The protein expression of LC3 was then examined by Western blotting. (<b>B</b>) After fixation, the distribution of TPC2-mRFP-EGFP was imaged under a confocal microscope. The number of RFP-positive signal dots per cells was quantified from the merged cell images. The scale bar indicates 20 μm. (n = 3, * <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001).</p> Full article ">