Fig 1.
PANC-1 cells differentiation and characterization with Activin-A.
(A) showed differentiation of Panc-1 cells subjected to differentiation using activin-A for 10 days. Figure shows bright field image of ILCC generated upon differentiation on day 10th at 10X magnification. Panels (A) showed Panc-1 ILCC immunostained on 10th day, positive for C-peptide, Insulin, and glucagon. Insulin was stained with TRITC labeled antibody showed red in color whereas c-peptide and glucagon were stained using FITC labeled antibodies showed green in color. (B) shows comparison of PANC-1 cells differentiation with Control SFM, Activin-A and Swertisin. Panc-1 cells cultured in complete media at day 0 which were then subjected to differentiation using SFM/ITS, Activin-A and Swertisin for 10 days. Bright field image of cells under differentiation for day 3rd, day 8th at 5X and 10X magnification respectively and dithizone stained ILCC on day 10th are shown. (C) shows comparative and qualitative insulin immunofluorescence signals in ILCC differentiated from SFM/ITS, Activin-A and Swertisin. Insulin is stained with Alexa-488 labeled antibody showed in red color and nucleus were counterstained with DAPI in blue. Pixel colocalization graphs shows co-localized distribution pattern of insulin and dapi fluorescence in differentiated ILCCs. (D) represents graph for quantification of insulin immunofluorescence per unit cytoplasm from immunostained ILCCs in Control SFM, Activin-A and Swertisin groups. Insulin signal stained with alexa-488 in Swertisin group compared to both SFM and activin-A group. Data was calculated from every single cell in three different frames per slide and expressed as mean ± SEM. *** and ** shows p value less than 0.001 and 0.01 in Swertisin group with respect to SFM and Activin-A group.
Fig 2.
Time course protein profiling and Fate of islet differentiation of Swertisin induced islet differentiation pathway from PANC-1 cells.
(A) represents immunoblotting of key parameters that indicate the movement of PANC-1 cells from precursor cells to endocrine islet-like cells, such as stem cell marker Nestin, pancreatic endocrine islet markers Ngn-3 and PDX-1. The activation of p38 MAP kinase to form phospho-P38 and replication marker Ki-67. Protein expression was quantified densitometrically from three independent experiment and expressed as Mean±SEM. *** shows p value <0.001 Vs Panc-1 undifferentiated cells and SFM/ITS. Immunoblotting of E cadherin and N Cadherin demonstrating fate of islet differentiation from undifferentiated PANC-1 cells to endocrine islet-like cells. Graph represents ratio of N-cadherin to E-cadherin depicting transition from precursor sate to differentiated state. Protein expression was quantified densitometrically from three independent experiments and expressed as Mean±SEM. (B) demonstrate characterization of Swertisin induced formation of islet-like clusters from PANC-1 cells in ten days time course. In this experiment shown, cells were harvested each day till ten days and immunoblotted for key parameters that indicate the movement of PANC-1 cells from precursor cells to endocrine islet-like cells in time dependent manner. Cells from control SFM/ITS and Swertisin, harvested from day 1 to 10 were probed for nestin, Ngn-3 PDX-1, phospho-P38 and E-cadherin. (C) shows short-term time course for key proteins implicated in islet differentiation pathway from Swertisin induced clusters. Immunoblotting of nestin, phospo-P38 and Ngn-3 in short-time course manner at 1, 3, 6, and 9 hours induction was performed. Lower images represent graphical representation of movement of protein expression in each group during 1 to 9 hours.
Fig 3.
Insulin fluorescence and Insulin content per unit cytoplasm quantification in Swertisin induced islet-like clusters derived from PANC-1 cells with activin-A and MAPKinase Inhibitor.
(A) shows fluorescent images for insulin expression in islet differentiation pathway inhibited using p38 MAP kinase inhibitor SB-203580 added in conjunction with Swertisin throughout ten days. The islet-like clusters were immunostained for insulin (green) on 10th day. Nuclear DNA was stained with DAPI (blue). (B) represents insulin content in islet like clusters after p-30 MAPK pathway inhibition. Graph represents insulin fluorescence quantification per unit cytoplasm in differentiated cells with and without inhibition of MAPK pathway using p38 MAP kinase inhibitor SB-203580 added in conjunction with Swertisin throughout ten days. Data is represented as Mean±SEM. *** and ** shows p value <0.001 and 0.01 Vs Activin-A and Swertisin alone groups respectively.
Fig 4.
Differentiation of mouse intra-islet progenitor cells and immunoblotting of key transcription factors and islet markers under differentiation with activin, swertisin and presence of p-38 MAPK inhibitor.
(A) depicts mIP cells cultured in complete media at day 0 which were then subjected to differentiation using activin-A and swertisin for 10 days. Bright field image shows, cells under differentiation on 8th day at 20X magnification, and dithizone stained clusters on day 10th. A fluorescent image represents immunostaining for insulin (green) and glucagon (red) in clusters from SFM/ITS, activin-a and swertisin groups. DAPI was used as nuclear stain. (B) shows immunoblotting of E-cadherin, N-cadherin, Ngn-3, P-p38, Native p-38 MAP kinase pathway proteins in presence and absence of MAP kinase pathway inhibitor SB203580. Ponceau S stain blot was shown as loading control. (C) shows immunoblotting of key differentiation pathway parameters that indicate the conversion of Mouse intra-islet progenitor cells into islet like clusters. Key transcription factors and MAP Kinase pathway proteins like P-p38, Erk1/2 Ngn-3, Pax-4, and Smad proteins under differentiation were monitored. Beta actin was used as loading control.
Fig 5.
In-vivo analysis of molecular mechanism by Swertisin differentiation in Ppx mice model.
(A) confocal images from regenerating pancreas for assessment of various islet transcription factors and signaling proteins of TGF-beta pathway from tissue of Ppx sham and Swertisin treated animals. Various markers like Ki67, Nestin, Ngn-3, CK19, p-smad-2, Smad-7, Insulin and Ck-19 were probed and analyzed. (B) showed western blot profile of stem cell markers, key transcription factors in islet differentiation pathway and cell death markers in PPx Sham and Swertisin treated animal pancreatic tissues.
Fig 6.
Proposed Mechanism of action of Swertisin mediated Islet Differentiation.
Panel shows proposed mechanistic action pathway for islet neogenesis being followed by Swertisin for new islet cell differentiation.