Figure S3. Independent replicates of B... | Rockefeller University Press

Figure S3.

$Independent replicates of B cell in vitro stimulation, confirmation of CRISPR-mediated NCF1 deletion, and impact of NCF1 deletion on p62 accumulation. (A) Splenic B cells cultured for 4 days on CD40L/BAFF-expressing feeder cells plus 10 ng/ml IL-21 and indicated dose of CpG. Percentage of CD138+IRF4+ shown. Each point represents the mean of technical replicates for each individual experimental animal. Bars, mean ± SEM. (B) Efficient CRISPR-mediated deletion of NCF1 in HBL1 cells by western blot. Also shown is a western blot for actin to normalized protein loading. Black arrow indicates the clone used for experiments. MW, molecular weight. (C) Western blot showing successful NCF1 deletion in primary human B cells. Actin staining was used to normalize protein loading. MW, molecular weight. (D) Fluorescent CpG maximum signal intensity in control and NCF1−/− HBL1 cells. Each point represents a single cell (n = 9–26 per condition). Independent replicate of data show in Fig. 6 B. (E) Pearson’s coefficient of EEA1 and CpG signal colocalization (above Costes threshold), masked by EEA1 staining after CpG stimulation for 15 and 30 min. Each point represents a single cell (n = 24–37/condition). Independent replicate of data show in Fig. 6 B. (F) Western blot of LC3-II in cytosolic fraction after CpG stimulation for 0–90 min, or after stimulation with rapamycin (R) and chloroquine (CQ) for 90 min to induce autophagy. Actin was used to normalize protein loading. MW, molecular weight. (G) Quantification of cytosolic LC3-II by densitometry (corrected for loading control). (H) Western blot of p62 in cytosolic fraction after stimulation with CpG for 0–90 min. Actin was used to normalize protein loading. MW, molecular weight. (I) Quantification of cytosolic p62 levels by densitometry (corrected for loading control). (B, D, G, and I) Bars show mean ± SEM of each individual experiment. Data were generated from two (A), four (G), five (I) independent experiments. (A, D, F, and H) *, P < 0.05; ***, P < 0.001 by two-tailed Student’s t test. Source data are available for this figure: SourceData FS3.$

Independent replicates of B cell in vitro stimulation, confirmation of CRISPR-mediated NCF1 deletion, and impact of NCF1 deletion on p62 accumulation. (A) Splenic B cells cultured for 4 days on CD40L/BAFF-expressing feeder cells plus 10 ng/ml IL-21 and indicated dose of CpG. Percentage of CD138⁺IRF4⁺ shown. Each point represents the mean of technical replicates for each individual experimental animal. Bars, mean ± SEM. (B) Efficient CRISPR-mediated deletion of NCF1 in HBL1 cells by western blot. Also shown is a western blot for actin to normalized protein loading. Black arrow indicates the clone used for experiments. MW, molecular weight. (C) Western blot showing successful NCF1 deletion in primary human B cells. Actin staining was used to normalize protein loading. MW, molecular weight. (D) Fluorescent CpG maximum signal intensity in control and NCF1^−/− HBL1 cells. Each point represents a single cell (n = 9–26 per condition). Independent replicate of data show in Fig. 6 B. (E) Pearson’s coefficient of EEA1 and CpG signal colocalization (above Costes threshold), masked by EEA1 staining after CpG stimulation for 15 and 30 min. Each point represents a single cell (n = 24–37/condition). Independent replicate of data show in Fig. 6 B. (F) Western blot of LC3-II in cytosolic fraction after CpG stimulation for 0–90 min, or after stimulation with rapamycin (R) and chloroquine (CQ) for 90 min to induce autophagy. Actin was used to normalize protein loading. MW, molecular weight. (G) Quantification of cytosolic LC3-II by densitometry (corrected for loading control). (H) Western blot of p62 in cytosolic fraction after stimulation with CpG for 0–90 min. Actin was used to normalize protein loading. MW, molecular weight. (I) Quantification of cytosolic p62 levels by densitometry (corrected for loading control). (B, D, G, and I) Bars show mean ± SEM of each individual experiment. Data were generated from two (A), four (G), five (I) independent experiments. (A, D, F, and H) *, P < 0.05; ***, P < 0.001 by two-tailed Student’s t test. Source data are available for this figure: SourceData FS3.