Mechanism of trypsin-mediated differentiation of pancreatic progenitor cells into functional islet-like clusters

Table 1 Primers of polymerase chain reaction for islet specific genes

Name	Sequences
Insulin-F	5’-GGAACGAGGCTTCTTCTACACA-3’
Insulin-R	5’-TGTTCCACAATGCCACGCTTCT-3’
Glucose-F	5’-ACATTCACCAGTGACTACAGCA-3’
Glucose-R	5’-GGCAGCTTGGCCTTCCAAATAA-3’
PAX6-F	5’-AGGCTCAAATGCGACTTCAGCT-3’
PAX6-R	5’-TGTTGCTGGCCTGTCTTCTCTGAT-3’
SOX9-F	5’-GGCAAGCTCTGGAGACTTCTG-3’
SOX9-R	5’-CCCGTTCTTCACCGACTTCC-3’
β-actin-F	5’-ATGAAGTGTGACGTGGACATCC-3’
β-actin-R	5’-CAGGAGGAGCAATGATCTTGATC-3’

PAX6: Paired box 6; SOX9: SRY-box transcription factor 9.

Table 2 Polymerase chain reaction procedure

Step	Temperature (°C)	Time	Number of cycles
Initial denaturation	94	5 minutes	1
Denaturation	94	30 seconds	35
Annealing	50-60	30 seconds
Extension	72	30 seconds/kb
Final extension	72	10 minutes	1
Cooling or holding	4	∞	1

Table 3 Antibody for immunofluorescence

Name	Cat and suppliers
Insulin	GB13121, Servicebio, Hubei Province, China
Glucagon	SAB4501137, Sigma-Aldrich, MO, United States
CSNK1G3	CSB-PA896549 LAOIHU, Cusabio, Hubei Province, China
E-cadherin	sc-8426, Santa Cruz, TX, United States

CSNK1G3: Casein kinase 1 gamma 3.

Table 4 Single-guide RNA for knockout

Name	Sequences
sgRNA-F2RL1-F	5’-caccgGTCTGCTTCACGACATACAA-3’
sgRNA-F2RL1-R	5’-aaacCTTGTATGTCGTGAAGCAGACc-3’
sgRNA-CDH1-F	5’-caccgCTACGGTTTCATAACCCACA-3’
sgRNA-CDH1-R	5’-aaacTGTGGGTTATGAAACCGTAGc-3’

CDH1: E cadherin; F2RL1: F2R like trypsin receptor 1; SgRNA: Single-guide RNA.

Table 5 Antibody for western blotting

Name	Cat and suppliers	Dilution ratios
Phospho-α-E-catenin (Ser655/Thr658)	13231, Cell Signaling Technology, Danvers, MA, United States	1:1000
Phospho-α-E-catenin (Ser641)	21330-1, Cell Signaling Technology, MA, United States	1:1000
Alpha-E-catenin	12831-1-AP, Proteintech, Hubei Province, China	1:1000
β-actin	A3854, Sigma-Aldrich, St Louis, MO, United States	1:50000
LRP5	24899-1-AP, Proteintech	1:1000
CK1α	ab206652, Abcam, Cambridge, United Kingdom	1:2000
CK1ε	ab270997, Abcam	1:2000
CK1γ3	df10135, Affinity, Jiangsu Province, China	1:1000
E-cadherin	20874-1-AP, Proteintech	1:2000
GAPDH	2118, Cell Signaling Technology	1:3000

CK: Casein kinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LRP5: Low-density lipoprotein receptor-related protein 5.