Fig. 1.
Unfolding protein response pathway scheme. ER – endoplasmic reticulum; PERK – protein kinase RNA-like ER kinase; eIF2α – eukaryotic translation initiation factor 2α; p-eIF2α – phosphorylated eukaryotic translation initiation factor 2α; GRP78 – glucose-regulated protein 78; ATF4 – activating transcription factor 4; CHOP – CCAAT/enhancer binding protein homologous protein
Table 1.
Gene Ontology knowledge base sets selected for the analysis
| Gene set | Species |
|---|---|
| GOBP_REGULATION_OF_PERK_MEDIATED_UNFOLDED_PROTEIN_RESPONSE | human |
| GOBP_REGULATION_OF_PERK_MEDIATED_UNFOLDED_PROTEIN_RESPONSE | mouse |
| HALLMARK_UNFOLDED_PROTEIN_RESPONSE | human |
| HALLMARK_UNFOLDED_PROTEIN_RESPONSE | mouse |
| REACTOME_UNFOLDED_PROTEIN_RESPONSE_UPR | human |
| REACTOME_UNFOLDED_PROTEIN_RESPONSE_UPR | mouse |
| WP_PHOTODYNAMIC_THERAPYINDUCED_UNFOLDED_PROTEIN_RESPONSE | human |
| WP_UNFOLDED_PROTEIN_RESPONSE | human |
Table 2.
Antibody list showing the percentage of protein identity between humans and dogs
| Protein | Clone | Supplier and catalogue No. | Dilution used in the study | % homology* |
|---|---|---|---|---|
| Anti-gamma H2AX | 9F3 | Abcam, Cambridge, UK, ab26350 | 1 : 1,000 in 3% BSA in TBS-T | 99.17 |
| Phospho-elF2α (Ser51) | 119A11 | Cell Signaling Technologies, Danvers, MA, USA, 3597 | 1 : 1,000 in 3% BSA in TBS-T | 96.07 |
| eIF2α | D-3 | Santa Cruz Biotechnology, Dallas, TX, USA, sc-133132 | 1 : 1,000 in 3% BSA in TBS-T | 96.07 |
| DDIT3/CHOP/GADD153 | B-3 | Santa Cruz Biotechnology, sc-7351 | 1 : 500 in 3% BSA in TBS-T | 92.3 |
| CHOP | L637F | Cell Signaling Technologies, 2895 | 1 : 1,000 in 3% BSA in TBS-T | 92.3 |
1* – homology according to the basic local alignment search tool for protein sequences; H2AX – histone family member X; eIF2α – eukaryotic translation initiation factor 2α; DDIT3 – DNA-damage–inducible transcript 3; CHOP – CCAAT/ enhancer binding protein homologous protein; GADD153 – growth-arrest and DNA-damage–inducible gene 153; BSA – bovine serum albumin; TBS-T – tris-buffered saline with Tween 20
Fig. 2.
Relative gene contents of the unfolded protein response (UPR) pathway and other pathways among genes expressed in the CLBL-1 B-cell lymphoma and GL-1 B-cell leukaemia canine cell lines (A) and relative expression of principal genetic components of the UPR pathway normalised to ACTB gene expression (B). ATF4 – activated transcription factor 4; EIF2A – eukaryotic translation initiation factor 2α; HSPA5 – heat-shock 70 kDa protein 5; DDIT3 – DNA-damage–inducible transcript 3; EIF2AK3 – eukaryotic translation initiation factor 2α kinase 3
Fig. 3.
Expression of unfolded protein response (UPR) proteins by canine lymphoma (CLBL-1 – B-cell lymphoma) and leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) cell lines treated with thapsigargin in order to induce endoplasmic reticulum stress (A). Expression levels were measured by Western blot and densitometry and the densitometric quantification of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) was normalised to β-actin. Fragment of human and canine gene and eIF2α protein sequences surrounding residue 51, showing the conserved serine (phosphorylation site when activation of the protein takes place) (B). CHOP – CCAAT/EBP enhancer binding protein homologous protein; GADD153 – growth-arrest and DNA-damage–inducible gene 153; cc – control; tg – thapsigargin; eto – etoposide; bp – base pairs; aa – amino acids
Fig. 4.
Expression of CCAAT enhancer-binding protein homologous protein (CHOP) by canine lymphoma (CLBL-1 – B-cell lymphoma and CNK-89 – natural-killer-cell lymphoma), canine leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) and human osteosarcoma (U2OS) cell lines treated with thapsigargin and MG132 in order to induce endoplasmic reticulum stress; expression being detected by a murine (CHOP (growth-arrest and DNA-damage–inducible gene 153 (GADD153) clone B-3) and a human (CHOP clone L637F) antibody (A). Quantification was assessed by densitometry. (B) Murine–human species epitope differences between CHOP/GADD153 B-3 and CHOP L637F with indication of the greater homology at the protein level between dogs and humans than between dogs and mice. CHOP L637F antibody immunogen surrounds amino acid 158, circled in red. cc – control; tg – thapsigargin; eto – etoposide; aa – amino acid; Ala – alanine
Fig. 5.
Apoptosis in canine lymphoma (CLBL-1 – B-cell lymphoma and CNK-89 – natural-killer-cell lymphoma) and leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) cell lines treated with thapsigargin and MG132 to induce endoplasmic reticulum stress and stained with annexin V. The upper left quadrants represent necrotic cells, the bottom left quadrants the living cell population, the upper right quadrants late apoptotic events and the bottom right quadrants early apoptotic events (A). Apoptosis in the same cell lines after the same treatments indicated by caspase 3/7 activation, confirming no major increase in apoptosis (except for in the CLBL-1 cell line). The second peak corresponds to apoptotic cells which harbour an active form of caspase 3/7. The red values represent means ± standard deviation (B). cc – control; FITC – fluorescein isothiocyanate