Ecl plus

Cell extracts were prepared in RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland). Samples mixed in loading buffer (Beyotime, Jiangsu, China) were heated to 100 °C for 10 min, separated on SDS‐polyacrylamide gel by electrophoresis, and electrotransferred onto polyvinylidene difluoride membrane (PVDF) membrane (Bio‐Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies for 2 h at room temperature. ECL Plus (Roche) was used to visualize the signals on the membrane. To obtain cell extracts that we needed, we irradiated cancer cells with 10Gy and collected the mixture after 5 days, when amount of PGCCs occurred according to our previous observation, and named it ‘PGCC + RC’. We then collected cells extracts after 10 days, when Raju cells proliferated to form a clone, and named it ‘RC’.

Total protein was extracted with RIPA lysis buffer containing protease inhibitor cocktail (Roche). Western blot was performed as previously described [16 (link)]. The membranes were incubated with primary antibodies purchased from Cell Signaling Technology (MA, USA. HMGB1, 1:1000 dilution; PCNA, 1:1000 dilution; CC3, 1:1000 dilution; β-actin, 1:1000 dilution) overnight at 4 °C and then incubated with secondary antibodies (1:5000 dilution, Jackson Immuno Research, PA, USA) for 2 h at room temperature. ECL Plus (Roche, Basel, Switzerland) was used to visualize the signals on the membrane.
To detect secretory HMGB1, cell culture supernatants were collected at 0 h, 6 h, 12 h, 1 day, 2 day, 3 day, 4 day, and 5 day. Secretory HMGB1 was detected through Western blotting shown above. Gray value of the bands in the Western blot was analyzed using ImageJ software [17 ].