![Figure 4. Treatment with si[IGLCCR] triggers ER stress and NOXA-related apoptosis. (A) Gene expression studies were performed using cDNA from 3 paired specimens of ALMC1 cells treated with si[IGLCCR] or si[-] on the Illumina platform using the standard protocols for HumanHT-12 v4. Expression BeadChip and the BeadStudio suite of programs were used to calculate the expression values for probe sets (Illumina Inc.). A heatmap of the 99 genes whose expression differed >1.5-fold between si[-] control and si[IGLCCR]-treated samples by multiple hypothesis testing at P < .10 is shown in supplemental Figure 3, increasing in expression from top to bottom. PMAIP1 (NOXA) is indicated in the supplemental figure with 1.54-fold increased expression in cells treated with si[IGLCCR]. The 24 genes that were most upregulated in cells treated with si[IGLCCR] are shown in this figure. HSPA5 (GRP78) is upregulated 2.4-fold. CHOP (also known as DDIT3) is the most upregulated gene consistent with activation of a terminal ER stress response. (B) GO heatmap was generated with GeneAnswers package in Bioconductor and shows gene expression values and pathways involved for the regulated genes, indicating that the UPR, ERAD, and apoptotic pathways are notably involved.18 (C) In this IB of si[-] control and si[CHOP]-, si[IGLCCR]-, and si[IGLCCR+CHOP]-treated ALMC1 cells, there were reductions in CHOP in the si[CHOP] and double knockdown cells; whereas in the si[IGLCCR] and double knockdown cells there were increases in GRP78, IRE1α, NOXA, MCL-1, and cleaved poly ADP ribose polymerase. (D) In ALMC1 cells at 20 hours after treatment, reduction of IgL expression was associated with increased caspase 3/7 activity, whereas simultaneous reduction of IgL and NOXA was associated with substantially reduced caspase 3/7 activity. Cells in which NOXA alone was knocked down had on average 17% less caspase 3/7 activity than controls. Mean ± SD with P value obtained with 2-tailed unpaired Student t test (n = 9). (E) In this IB of si[-] control and si[NOXA]-, si[IGLCCR]-, and si[IGLCCR + NOXA]-treated ALMC1 cells, there were increases in GRP78, IRE1α, and phosphorylated PERK in si[IGLCCR]-treated cells; reductions in NOXA in the si[NOXA] and double knockdown cells; but no differences in the levels of MCL-1 with NOXA knockdown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/22/10.1182_blood-2013-10-535187/4/3440f4.jpeg?Expires=1735557975&Signature=k-uuynwvVh7ZdkPgKfodNNOI5FtXrztEmmPeOar~nalsqsmfMpktQs3a8O2PNAcC36b7yisyAP8HNS~1HImNHWqNRp4Y2dkOeLcpD5gEROVTMOl9hCZttUy6dWsa4kKmIRvlVUcERs7hlvgirdLlvtMwRTLRCt-aV0klWSVAePWlC~LbVHtpviJ6qQ87ppoZqgqll1GNJtvZra5h5JdTANKA7JlLM-5kTBL0ZJjOJYY10n58YrUDMZ7RQW1DjIUjndW13tMTsvf-FAPyo9j4J4xDPaLS2u6SmI4-X0lcZbNf8~BuJYxWBdATg6G75FXUnknlp0onLJpL~hWObhJkVg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Treatment with si[IGLCCR] triggers ER stress and NOXA-related apoptosis. (A) Gene expression studies were performed using cDNA from 3 paired specimens of ALMC1 cells treated with si[IGLCCR] or si[-] on the Illumina platform using the standard protocols for HumanHT-12 v4. Expression BeadChip and the BeadStudio suite of programs were used to calculate the expression values for probe sets (Illumina Inc.). A heatmap of the 99 genes whose expression differed >1.5-fold between si[-] control and si[IGLCCR]-treated samples by multiple hypothesis testing at P < .10 is shown in supplemental Figure 3, increasing in expression from top to bottom. PMAIP1 (NOXA) is indicated in the supplemental figure with 1.54-fold increased expression in cells treated with si[IGLCCR]. The 24 genes that were most upregulated in cells treated with si[IGLCCR] are shown in this figure. HSPA5 (GRP78) is upregulated 2.4-fold. CHOP (also known as DDIT3) is the most upregulated gene consistent with activation of a terminal ER stress response. (B) GO heatmap was generated with GeneAnswers package in Bioconductor and shows gene expression values and pathways involved for the regulated genes, indicating that the UPR, ERAD, and apoptotic pathways are notably involved.18 (C) In this IB of si[-] control and si[CHOP]-, si[IGLCCR]-, and si[IGLCCR+CHOP]-treated ALMC1 cells, there were reductions in CHOP in the si[CHOP] and double knockdown cells; whereas in the si[IGLCCR] and double knockdown cells there were increases in GRP78, IRE1α, NOXA, MCL-1, and cleaved poly ADP ribose polymerase. (D) In ALMC1 cells at 20 hours after treatment, reduction of IgL expression was associated with increased caspase 3/7 activity, whereas simultaneous reduction of IgL and NOXA was associated with substantially reduced caspase 3/7 activity. Cells in which NOXA alone was knocked down had on average 17% less caspase 3/7 activity than controls. Mean ± SD with P value obtained with 2-tailed unpaired Student t test (n = 9). (E) In this IB of si[-] control and si[NOXA]-, si[IGLCCR]-, and si[IGLCCR+ NOXA]-treated ALMC1 cells, there were increases in GRP78, IRE1α, and phosphorylated PERK in si[IGLCCR]-treated cells; reductions in NOXA in the si[NOXA] and double knockdown cells; but no differences in the levels of MCL-1 with NOXA knockdown.
