Figure 3. Loss of HMGB2 blunts SASP gene... | Rockefeller University Press

$Figure 3. Loss of HMGB2 blunts SASP gene expression while maintaining the senescence-associated cell growth arrest. (A) IMR90 cells were infected with retrovirus encoding oncogenic RAS alone or in combination with a lentivirus expressing an shRNA to the human HMGB2 gene (shHMGB2). Cells were selected for 3 d with 3 µg/ml puromycin. 4 d later, HMGB2 mRNA expression was determined. (B) Same as A, but both total cell lysates (TCL) and chromatin fractions were isolated, and HMGB2 protein expression was determined. Histone H3 and β-actin were used as internal loading controls. (C) Same as A, but IL1β, IL8, and IL6 mRNA expression was determined. (D) IMR90 cells were infected with retrovirus encoding oncogenic RAS alone or in combination with a Cas9-expressing lentivirus expressing a gRNA to HMGB2 (CRISPR). Cells were selected for 3 d with 3 µg/ml puromycin. 4 d later, the chromatin fraction was isolated, and HMGB2 protein expression was determined. Histone H3 was used as an internal loading control. (E) Same as D, but IL1β, IL8, and IL6 mRNA expression was determined. (A, C, and E) B2M was used as an internal control. (F) Same as A, but SA-β-Gal staining was performed. Bars, 5 µm. (G) Quantification of F. (H) Same as A, but an equal number of cells were seeded into 6-well plates and stained with crystal violet 14 d later. (I) Quantification of H. Graphs shown are the mean and SEM of triplicates from a representative experiment that was independently repeated at least three times. *, P < 0.05 versus control; #, P < 0.05 versus RAS.$

Figure 3.

Loss of HMGB2 blunts SASP gene expression while maintaining the senescence-associated cell growth arrest. (A) IMR90 cells were infected with retrovirus encoding oncogenic RAS alone or in combination with a lentivirus expressing an shRNA to the human HMGB2 gene (shHMGB2). Cells were selected for 3 d with 3 µg/ml puromycin. 4 d later, HMGB2 mRNA expression was determined. (B) Same as A, but both total cell lysates (TCL) and chromatin fractions were isolated, and HMGB2 protein expression was determined. Histone H3 and β-actin were used as internal loading controls. (C) Same as A, but IL1β, IL8, and IL6 mRNA expression was determined. (D) IMR90 cells were infected with retrovirus encoding oncogenic RAS alone or in combination with a Cas9-expressing lentivirus expressing a gRNA to HMGB2 (CRISPR). Cells were selected for 3 d with 3 µg/ml puromycin. 4 d later, the chromatin fraction was isolated, and HMGB2 protein expression was determined. Histone H3 was used as an internal loading control. (E) Same as D, but IL1β, IL8, and IL6 mRNA expression was determined. (A, C, and E) B2M was used as an internal control. (F) Same as A, but SA-β-Gal staining was performed. Bars, 5 µm. (G) Quantification of F. (H) Same as A, but an equal number of cells were seeded into 6-well plates and stained with crystal violet 14 d later. (I) Quantification of H. Graphs shown are the mean and SEM of triplicates from a representative experiment that was independently repeated at least three times. *, P < 0.05 versus control; #, P < 0.05 versus RAS.

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