EMBO/soda-nel huggingface dataset:
Stress signaling in breast cancer cells induces matrix comp. that promote chemoresistant metastasis (2018)
Figure 3

Figure 3. JNK signaling promotes stem cell properties and invasiveness in breast cancer cells.

A Gene expression profile of MDA231-LM2 cells grown as oncospheres or as a monolayer. Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red.

B GSEA graph showing enrichment of JNK signature in MDA231-LM2 cancer cells grown as oncospheres. NES, normalized enrichment score.

C Immunoblot of phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in oncospheres from SUM159-LM1 cells. Tubulin was included as a loading control.

D GSEA of a basal-like breast cancer signature (Perou et al, 2000) induced in MDA231-LM2 oncospheres.

E Normalized enrichment score (GSEA) of mammary stem cell signatures in oncospheres from MDA231-LM2 cells. Signatures a-d are the same as described in Fig. 2E. *P < 0.05, ***P < 0.001. NS, not significant. For panels B, D and E, P values were determined by random permutation test.

F Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK. Values are mean from 10 wells per group ± SD.

G Oncosphere formation in SUM159-LM1 cells following treatment with JNKi or vehicle control. Values are mean from 10 wells per condition ± SD.

H Representative images of spheres from panel G. Scale bar, 100 μm.

I, J Oncosphere formation in JNKi-treated cancer cells from breast cancer patients (pleural effusions or ascites). (I) Quantification of oncospheres per well in 4 different patients. Values are mean from 10 wells ± SD. (J) Representative oncospheres from patient samples. Scale bar, 50 μm.

K Subcutaneous injection of limiting numbers of JNKi-treated MDA231-LM2 cells in NSG mice.

L Analysis of tumor initiation ability of vehicle- and JNKi-treated cells from experiment described in panel (K); n = 3 mice per group, two implantations per mouse.

M Matrigel invasion assay of MDA231-LM2 cells treated with JNKi. Left, examples of invading cancer cells stained with crystal violet (arrows). Scale bar, 50 μm. Right, quantification of invading cells per field of view (FOV) showing mean from 8 fields ± SEM.

For panels F, G, I, and M, **P < 0.01; ***P < 0.001; ****P < 0.0001. P values were determined by a two-tailed Mann-Whitney test.
Stress signaling in breast cancer cells induces matrix comp. that promote chemoresistant metastasis (2018)
Figure 5

Figure 5. SPP1 and TNC are induced by chemotherapy via JNK signaling.

A Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment.

B Venn diagram showing overlap between genes that were significantly (P < 0.05) induced more than 1.5-fold in response to paclitaxel treatment of MDA231-LM2 cells and genes that were significantly (P < 0.05) induced in MDA231-LM2 cells after formation of oncospheres. Significant fold changes were determined by two-sample empirical Bayer tests with Benjamini-Hochberg correction for multiple testing. PAX.

C GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells. Signatures a-d are the same as in Fig. 2E. *P < 0.05, ***P < 0.001. NS, not significant. P values were determined by random permutation test.

D Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm.

E Quantification of cancer cells expressing p-c-Jun in panel D. Values are mean from three mice ± SEM. Nine metastatic foci were analyzed per mouse. P value was determined by a two-tailed Mann-Whitney test.

F Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.

G, H SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX or doxorubicin (DOXO).

I SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours.

J SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi.

For panels G-J, values represent the mean of qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells. P values were determined by a Mann-Whitney test.
Stress signaling in breast cancer cells induces matrix comp. that promote chemoresistant metastasis (2018)
Figure 6

Figure 6. SPP1 and TNC promote chemoresistance.

A Experimental treatment schedule of mice after cancer cell implantation in the mammary gland to address the role of SPP1 in resistance to PAX.

B Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors. Each value represents the mean ± SEM. ****P < 0.0001.

C Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. 10 fields per lung section were quantified. Each value represents the mean ± SEM.

D Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors. Boxes represent median with upper and lower quartiles. Whiskers show maximum and minimum. P value was determined by two-tailed Mann-Whitney test.

E Lung metastasis was quantified in mice that were implanted with MDA231-LM2 tumors and subjected to AC chemotherapy. Each value represents the mean ± SEM; n = 5 mice per group. Ten random fields per lung section were analyzed for percentage of metastatic area.

F Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E. Metastases were determined by expression of human vimentin (arrows). Scale bar, 100 µm.

G Treatment schedule after cancer cell implantation to address the role of TNC in PAX resistance in a xenograft mouse model for breast cancer metastasis to lungs. Mice were implanted with MDA231-LM2 cells expressing either control shRNA (control) or a TNC shRNA (shTNC), treated with paclitaxel or vehicle.

H Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P < 0.0001.

I Lung metastases quantified in tumor bearing mice from panel (H).

J Representative histology of human vimentin-positive lung metastases (arrows) from the experiment described in panels G-I. Scale bar, 100 µm.

P values in panels B, C, D, E, H and I were determined by two-tailed Mann-Whitney test.
Stress signaling in breast cancer cells induces matrix comp. that promote chemoresistant metastasis (2018)
Figure 7

Figure 7. Inhibition of JNK signaling sensitizes mammary tumors and metastases to chemotherapy.

A Experimental scheme to determine whether JNK signaling promotes resistance to chemotherapy. MDA231-LM2 or SUM159-LM1 cells were implanted bilaterally into the fourth mammary fat pads of NSG mice. Treatment with JNKi was initiated at day 5 post implantation and repeated every three days; paclitaxel chemotherapy was started at day 10 post implantation and repeated every five days.

B Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001.

C Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel.

D Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm.

E Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM. For panels B, C and E, P values were determined by two-tailed Mann-Whitney test.

F GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial (GSE16446), stratified according to pathological response to neoadjuvant chemotherapy. NES, normalized enrichment score. pCR, pathological complete response. P value was determined by random-permutation test.

G, H Kaplan-Meier analyses of chemotherapy-treated breast cancer patients associating JNK signature (JNK-S) with metastasis-free survival (TOP trial data set, JNK-S low n = 54 patients; JNK-S high n = 53 patients (G)) or overall survival (METABRIC data set, JNK-S low n = 135 patients; JNK-S high n = 135 patients (H)). HR, hazard ratio. P values were determined by log-rank test.

I Model illustrating the link between stress-induced ECM, stem cell and wound healing network and metastatic progression and therapy resistance in breast cancer.

EXPANDED VIEW FIGURE LEGENDS
Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury (2013)
Figure 1

The recruitment of ubiquitin and LC3 to GFP‐Gal3‐positive damaged lysosomes. (A–C) Atg7+/+ and Atg7–/– MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.
Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury (2013)
Figure 2

Decrease in the number of GFP‐Gal3 puncta is dependent on time and autophagy. (A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.
Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury (2013)
Figure 3

tfGal3 GFP signal in puncta attenuates in an autophagy‐dependent manner. (A) Diagram of the primary structure of tandem fluorescence‐tagged Galectin‐3 (tfGal3). (B) Schematic diagram of the fate of tfGal3 recruited to damaged lysosomes. (C–E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G‐Count (D). Then, the percent of GFP+RFP+ tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm. (F–H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B‐mutant (H) cells was quantified by G‐Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.
Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury (2013)
Figure 4

CLEM analysis of mSt‐Gal3‐ and GFP‐LC3‐associated membranes. (A–F) HeLa cells stably expressing GFP‐LC3 were transfected with mStrawberry‐Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B–F). Green: LC3; magenta: Gal3; blue: DAPI. (G–I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.
Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury (2013)
Figure 5

Autophagy‐deficient renal tubules exhibit severe injury and dysfunction under hyperuricaemia. (A) Plasma creatinine and urea nitrogen in Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. (B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4–7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C). (D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm. (E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm. (F) Model of autophagic clearance of damaged lysosomes.Source data for this figure is available on the online supplementary information page.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 1

TLR8 ligands induce autophagy in human macrophages.

Macrophages were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Immunoblot of co-immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β-actin as loading control. (B) Cells were incubated with 10 µg/mL pepstatin A for 4 h prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin. (C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 6. (D) Immunoblots of SQSTM1 using antibody to SQSTM1 or β-actin. * p<0.05.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 2

ssRNA40 and CL097 induce autophagy in macrophages through TLR8.

Macrophages transduced with non-specific scrambled shRNA (shNS) or TLR8 shRNA (shTLR8) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Cells were lysed and immunoblot performed with antibodies to TLR8 and β-actin. (B) Immunoblots of LC3B isoforms using antibody to LC3B or β-actin. (C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images from three donors are shown. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 4. * p<0.05.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 3

ssRNA40- and CL097-induced autophagy in macrophages is mediated through CYP27B1 and the VDR.

(A–B) Macrophages were treated for 24 h with CL097, ssRNA40 or ssRNA41. (A) Top, qRT-PCR for CYP27B1 after 6 h. Bottom, immunoblots of CYP27B1 using antibody to CYP27B1 or β-actin. (B) Top, qRT-PCR for VDR after 6 h. Bottom, immunoblots of VDR using antibody to VDR or β-actin. (C–D) Macrophages transduced with non-specific scrambled shRNA (shNS), CYP27B1 shRNA (shCYP27B1) or VDR shRNA (shVDR) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (C) Left, immunoblots performed with antibodies to CYP27B1, VDR and β-actin. Right, immunoblots of LC3B isoforms using antibody to LC3B or β-actin. (D) Cells were fixed and permeabilized then stained with antibody to LC3B. Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s.e.m., n = 4. (E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or β-actin. Bar charts are reported as mean ± s.e.m., n = 4. * p<0.05.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 4

CYP27B1 induction in response to TLR8 ligands is CEBPB-dependent.

Macrophages transduced with non-specific scrambled shRNA (shNS) or CEBPB shRNA (shCEBPB) were treated for 24 h with CL097, ssRNA40, or ssRNA41. (A) Cells were lysed and immunoblot performed with antibodies to CEBPB and β-actin. (B) Immunoblots of CYP27B1 expression using antibody to CYP27B1 or β-actin.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 5

TLR8 agonists inhibit HIV replication.

Macrophages were incubated with (A) ssRNA40 or (B) CL097 for 24 h before infection with HIV for 3 h. Cells were then washed and incubated with ssRNA40 or CL097 for 10 d. Extracellular release of HIV p24 antigen into the cell supernatant at days 0, 3, 5, 7, 10 was detected by ELISA. Results are reported as mean ± s.e.m., n = 5. * p<0.05.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 6

TLR8 agonist inhibition of HIV dependent upon a functional vitamin D pathway.

(A) Macrophages transduced with non-specific scrambled shRNA (shNS), TLR8 shRNA (shTLR8), CYP27B1 shRNA (shCYP27B1), or VDR shRNA (shVDR) were treated with CL097 for 24 h before infection with HIV with continuous CL097 treatment. Top, immunoblot analysis performed using antibodies raised to TLR8, CYP27B1, VDR and β-actin. Bottom, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4. (B) Macrophages were incubated with ssRNA40 or CL097 in the presence of 45 or 100 nmol/L 25D3 for 24 h before infection with HIV. Cells were then washed and incubated with 25D3 and ssRNA40 or CL097 for 10 d Extracellular release of HIV p24 antigen was detected by ELISA. Results are reported as mean ± s.e.m., n = 6. * p<0.05.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 7

TLR8-mediated inhibition of HIV is autophagy dependent.

(A–B) Macrophages transduced with BECN1 shRNA (shBECN1) (A) or ATG5 shRNA (shATG5) (B) were incubated with CL097 for 24 h before infection with HIV with continuous CL097 treatment for 10 d. Right, immunoblot analysis performed using antibodies raised to BECN1, ATG5 and β-actin. Left, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4. (C) Macrophages were pretreated with bafilomycin A1 (Baf A1), SID 26681509 (SID) or vehicle control before incubation with CL097 or ssRNA40 for 24 h before infection with HIV. Cells were then washed and incubated with TLR8 agonists and inhibitors for 10 d and ELISA performed for extracellular HIV p24 antigen release over 10 d. Results are reported as mean ± s.e.m., n = 5. * p<0.05; # p<0.001.
Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (2012)
Figure 8

Inhibition of HIV by TLR8 agonists is CAMP and autophagy dependent.

(A) Macrophages were treated with CL097, ssRNA40, ssRNA41 for 6 h after which qRT-PCR for CAMP was performed. Results are reported as mean ± s.e.m., n = 4. (B) Macrophages were treated for 24 h with CL097 or ssRNA40 in the presence of 45 nmol/L or 100 nmol/L 25D3. Left, qRT-PCR for CAMP performed after 6 h. Results are reported as mean ± s.e.m., n = 4. Right, after 24 h cells were stained with anti-CAMP antibodies and analyzed by flow cytometry. Representative histograms from three donors are shown. (C) Macrophages transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) were incubated with CL097, rapamycin (Rapa) or vehicle control for 24 h. Top, immunoblot analysis performed using antibodies raised to CAMP and β-actin. Bottom, immunoblots of LC3B isoforms using antibody to LC3B or β-actin. (D) After 24 h treatment with CL097 or ssRNA40, macrophages from part C were also washed and infected with HIV and incubated with CL097, ssRNA40 or vehicle control for 10 d. ELISA was performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 6. * p<0.05.
TECRL, a new life-threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT (2016)
Figure 2

Figure 2. Spatiotemporal, tissue and sub-cellular expression analysis of TECRL.

(A-H) Expression of Tecrl in mouse development. Expression is not observed in (A) the cardiac crescent at E7.5 but is prominent in (B) the inflow tract (ift) region at E8.5, particularly in (C) the left sinus venosus (lsv). (D) At E9.5, Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract. (E-G) From E10 onwards, Tecrl is expressed in the somites. (H) At E14.5, Tecrl expression is present in the entire myocardium; [atrium (a), ventricle (v), neural tube (nt), lung (lu), right atrium (ra), left atrium (la), right ventricle (rv) and left ventricle (lv)]. (I) mRNA expression analyses of TECR and TECRL by RT-qPCR in human tissues demonstrates preferential expression of Tecrl in the heart; [Bone marrow (Bone M.), heart, skeletal muscle (Sk.Muscle), uterus, liver, spleen, thymus, thyroid, prostate, brain, lung, small intestine (Small I.) and colon] (J) Localization of MYC-Tecrl in COS-1 cells. The accumulation of MYC-Tecrl is perinuclear consistent with it being localized to the ER. (K-M) Co-localization of MYC-Tecrl and the ER marker Calnexin in H10 cells. Nuclei stained with DAPI. Scale bar: 10 μm.