Figure 2.
Functional evaluation of patient fibroblast cell lines shows hypomorphic nature of the c.3624C>T variant. (A) Western blot of the indicated cell lines with and without MMC treatment. Weak monoubiquitination of FANCD2 is observed in cells RA2349EH (from individual 2), and RA2565EH (from individual 3) in response to MMC. RA3087EH cells (individual with FA with biallelic deletions in FANCA) cells were used as negative controls. BJ cells were used as positive control. (B) Quantification of FANCD2 foci in the indicated cell lines. Proficiency of FANCD2 localization to sites of damage is suppressed in RA2349EH and RA2565EH in response to MMC but is higher than in FANCA-null cells (RA3087EH). Unpaired t test was used to test for significance between untreated and MMC-treated fibroblasts. Also see supplemental Figure 1. (C) Intermediate level of survival is observed for RA2349EH and RA2565EH cells in response to increasing concentrations of MMC. Cell survival was calculated relative to untreated cells. Error bars represent standard deviation (SD) of an experiment done in triplicate. Data from a representative experiment are shown with the SD showing the range of technical replicates. RA3087EH complemented with empty vector (EV) were used as a negative controls. RA3087EH complemented with wildtype FANCA (WT-FANCA) were used as a positive control. Statistical analysis was performed using data from 2 independent experiments, each with 3 technical replicates. Adjusted P values from multiple comparison 2-way analysis of variance analysis of differences in survival between RA3087EH+EV (FANCA null) and the other cell lines were P < .0001 at 5 and 10 nM of MMC for all 3 cell lines. P < .0001, P < .0057, and P < .0002 for WT, RA2349, and RA2565, respectively, at 25 nM MMC. At 50 nM MMC, the P value was significant (P < .0001) for the WT cell line only. (D) Cell cycle analysis without MMC treatment (blue line) or after exposure to 25 nM MMC (red line) in the indicated cell lines. Three independent experiments were performed and the average percentages of cells in the late S/G2 cell cycle stage are indicated. (E) Western blot analysis of FANCA in the indicated cell lines. RA2349EH and RA2565EH cells showed expression of residual endogenous FANCA protein (indicated by an asterisk “∗”).

Functional evaluation of patient fibroblast cell lines shows hypomorphic nature of the c.3624C>T variant. (A) Western blot of the indicated cell lines with and without MMC treatment. Weak monoubiquitination of FANCD2 is observed in cells RA2349EH (from individual 2), and RA2565EH (from individual 3) in response to MMC. RA3087EH cells (individual with FA with biallelic deletions in FANCA) cells were used as negative controls. BJ cells were used as positive control. (B) Quantification of FANCD2 foci in the indicated cell lines. Proficiency of FANCD2 localization to sites of damage is suppressed in RA2349EH and RA2565EH in response to MMC but is higher than in FANCA-null cells (RA3087EH). Unpaired t test was used to test for significance between untreated and MMC-treated fibroblasts. Also see supplemental Figure 1. (C) Intermediate level of survival is observed for RA2349EH and RA2565EH cells in response to increasing concentrations of MMC. Cell survival was calculated relative to untreated cells. Error bars represent standard deviation (SD) of an experiment done in triplicate. Data from a representative experiment are shown with the SD showing the range of technical replicates. RA3087EH complemented with empty vector (EV) were used as a negative controls. RA3087EH complemented with wildtype FANCA (WT-FANCA) were used as a positive control. Statistical analysis was performed using data from 2 independent experiments, each with 3 technical replicates. Adjusted P values from multiple comparison 2-way analysis of variance analysis of differences in survival between RA3087EH+EV (FANCA null) and the other cell lines were P < .0001 at 5 and 10 nM of MMC for all 3 cell lines. P < .0001, P < .0057, and P < .0002 for WT, RA2349, and RA2565, respectively, at 25 nM MMC. At 50 nM MMC, the P value was significant (P < .0001) for the WT cell line only. (D) Cell cycle analysis without MMC treatment (blue line) or after exposure to 25 nM MMC (red line) in the indicated cell lines. Three independent experiments were performed and the average percentages of cells in the late S/G2 cell cycle stage are indicated. (E) Western blot analysis of FANCA in the indicated cell lines. RA2349EH and RA2565EH cells showed expression of residual endogenous FANCA protein (indicated by an asterisk “∗”).

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