Figure 3.
Figure 3. Gene expression changes in CD28- vs CD86-silenced cells are consistent with regulation of both distinct and common pathways, including expression of IRF4. (A) Gene set enrichment analysis showing upregulated (top) and downregulated (bottom) gene sets in shCD28- (blue) and shCD86-treated (red) myeloma cells. For each gene set, the enrichment score is shown above the ranked change in gene expression, where genes that overlap the gene set are denoted by blue and red ticks for shCD28 and shCD86, respectively. (B) qRT-PCR showing IRF4 mRNA levels when CD28 or CD86 was silenced in MM.1s or 8226 cells. (C) Representative western blots showing IRF4 levels with silencing of CD28 or CD86 in cell lines indicated at different time points. (D) Integrin levels were measured via qRT-PCR (ITGB7 and ITGB1; left). Representative histograms showing ITGβ1 and ITGβ7 on day 4 after shRNA treatment (right). For qRT-PCR, all data are normalized to β-actin as an endogenous control and then compared relative to mRNA levels in pLKO.1 empty vector control. RNA was extracted at day 3 postinfection. Data shown are mean ± SEM of at least 3 independent experiments. Histograms showing surface levels of indicated molecules. Gray histograms at left represent unstained or isotype controls. Flow cytometry data shown are from day 4 postinfection with lentiviral vectors. Flow cytometry and western blot data shown are representative of at least 3 independent experiments. (E) Cell adhesion 3 days postinfection with lentivirus containing the indicated shRNA. Myeloma cells stained with calcein AM were cocultured with HS-5 cells for 2 hours. Fluorescence was measured (485/528 emission/excitation) with BioTek Synergy H1 multiwell plate reader, and data are presented as fluorescence relative to pLKO.1 controls (mean ± SEM). Prewash readings were taken to ensure similar numbers of live cells were added to each well. All samples were plated in triplicate. Data are mean of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GX, gene expression changes; RFU, relative fluorescence unit.

Gene expression changes in CD28- vs CD86-silenced cells are consistent with regulation of both distinct and common pathways, including expression of IRF4. (A) Gene set enrichment analysis showing upregulated (top) and downregulated (bottom) gene sets in shCD28- (blue) and shCD86-treated (red) myeloma cells. For each gene set, the enrichment score is shown above the ranked change in gene expression, where genes that overlap the gene set are denoted by blue and red ticks for shCD28 and shCD86, respectively. (B) qRT-PCR showing IRF4 mRNA levels when CD28 or CD86 was silenced in MM.1s or 8226 cells. (C) Representative western blots showing IRF4 levels with silencing of CD28 or CD86 in cell lines indicated at different time points. (D) Integrin levels were measured via qRT-PCR (ITGB7 and ITGB1; left). Representative histograms showing ITGβ1 and ITGβ7 on day 4 after shRNA treatment (right). For qRT-PCR, all data are normalized to β-actin as an endogenous control and then compared relative to mRNA levels in pLKO.1 empty vector control. RNA was extracted at day 3 postinfection. Data shown are mean ± SEM of at least 3 independent experiments. Histograms showing surface levels of indicated molecules. Gray histograms at left represent unstained or isotype controls. Flow cytometry data shown are from day 4 postinfection with lentiviral vectors. Flow cytometry and western blot data shown are representative of at least 3 independent experiments. (E) Cell adhesion 3 days postinfection with lentivirus containing the indicated shRNA. Myeloma cells stained with calcein AM were cocultured with HS-5 cells for 2 hours. Fluorescence was measured (485/528 emission/excitation) with BioTek Synergy H1 multiwell plate reader, and data are presented as fluorescence relative to pLKO.1 controls (mean ± SEM). Prewash readings were taken to ensure similar numbers of live cells were added to each well. All samples were plated in triplicate. Data are mean of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GX, gene expression changes; RFU, relative fluorescence unit.

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