Isopropanol

Total DNA was extracted with cetyl-trimethyl-ammonium bromide (CTAB) (Sangon Biotech, China). Briefly, leaf samples were first ground and mixed with CTAB solutions containing 2% β-mercaptoethanol (Sigma, Saint Louis, MO, USA). Next, these samples were incubated at 65 °C and then trichloromethane (Sangon Biotech, China) was added. Samples were then centrifuged, upper phase was collected and mixed with isopropanol (Sangon Biotech, China). DNA was obtained by centrifugation and washing with 75% ethanol solution. Total RNA was extracted using TRIzol (Ambion, Austin, TX, USA). Samples were first ground and mixed with TRIzol. Trichloromethane was added and samples were then centrifuged. Supernatants were collected and mixed with isopropanol. After washing with ethanol solution, RNA was obtained by centrifugation. For the quantification of begomoviruses and betasatellites in DNA samples, SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA) were used. For the analysis of gene expression level of AV1 and βC1, cDNA was synthesized using Evo M-MLV RT Kit with gDNA Clean for qPCR (Accurate Biology, China). QPCR was then performed as mentioned above. Primers are listed in Table 1.

Ethylene diamine tetraacetic acid (EDTA), cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), Tris-HCl, NaCl, urea, Na₂CO₃, NaHCO₃, sodium citrate (C₆H₅Na₃O₇), guanidine isothiocyanate (C₂H₆N₄S), sodium N-dodecanoylsalcosinate (C₁₅H₁₀NO₄Na), β-mercaptoethanol, alcohol, isoamyl alcohol, and isopropanol were purchased from Sangon Biotech (Shanghai, China). HAuCl₄ was purchased from Sinopharm (Shanghai, China). Bovine serum albumin (BSA), sucrose, and alginate were obtained from Meilunbio Biotech (Dalian, China). Absorbent pads (CH27) and sample pads (SB06) were purchased from KinBio Biotech (Shanghai, China). The gold absorbent pad (GL0194), PVC backing plate (DB-6), and nitrocellulose membrane (Millipore 135) were obtained from Jiyi Biotech (Shanghai, China). PCR Master Max and the anti-5-FAM antibody were obtained from BBI Biotech (Shanghai, China). Streptavidin and biotin-BSA were obtained from Solarbio Biotech (Beijing, China).

Sodium alginate (SA), N,N’-methylenebisacrylamide (MBAA), N,N,N,N-tetramethylethylenediamine (TEMED), and calcium sulfate dihydrate (CaSO₄·H₂O) were purchased from Adamas. Ammonium persulfate (APS), (3-aminopropyl)triethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (TMSPMA), 1,6-hexamethylenediamine (HMDA) and benzophenone (BP) were purchased from Aladdin. Potassium chloride was purchased from Rhawn. Acrylamide (AM) was purchased from General Reagent. Glycerol and methanol were purchased from Damao. Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) was purchased from Bidepharm. Isopropanol was purchased from Sangon Biotech. Ethanol was purchased from Chemical Reagent. All chemicals were used as received without further purification.

The mature adipocytes were grown on a coverslip and fixed with 10% formalin (Guangzhou Dingguo Biology, Guangdong, China) at room temperature for 1 h. After fixation, the cells were washed once with 60% isopropyl alcohol (Sangon Biotech, Shanghai, China). Oil Red O solution (Sangon Biotech, Shanghai, China) was prepared according to the manufacturer’s instructions and added to the plate for 1 h. The coverslips were then visualized under a microscope. The cell culture plates were treated with isopropanol (Sangon Biotech, Shanghai, China), and lipid accumulation was determined according to absorbance at 510 nm.

One milliliter of TRI Reagent^® (Sigma, St. Louis, MO, USA) was applied to dissociate the specimens and cells. Isopropanol (Sangon Biotech, Shanghai, China) was used to precipitate the total RNA. After measuring the concentration of RNA with a spectrophotometer (Tiangen, Peking, China), 1000 ng of total RNA was reverse transcribed to cDNA, and then, 1 µl cDNA was employed to conduct quantitative PCR (polymerase chain reaction) following the instructions of the manufacture (GeneCopoeia, Rockville, MD, USA). The specific primers of CREB1, CCAR1, JNK1, GAPDH, miR-433 and snRNA U6 were also purchased from GeneCopoeia. The 2^-△△CT method was adopted to analyze the relative expression.