EMBO/soda-nel huggingface dataset:
Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation. (2015)
Figure 1

Copper-binding conformations and PrP octarepeat sequence mutations

A The structure of full-length PrP and derivative C1 and C2 fragments with an enlarged view of the cleavage sites is shown. The C2 (β) site was mapped in HpL3-4 cells by (Mange et al, 2004). Ragged C1 (α) cleavage sites in brain are shown (Chen et al, 1995), and a dotted arrow indicates that additional sites have been mapped rightward of this position in vitro (McDonald et al, 2014). Ragged N-terminal cleavage sites of PrP27-30 from prion-infected mouse brain are indicated by small arrows (Howells et al, 2008). The positions of antibody epitopes are also shown (residues in brackets). Grey shading reflects the minimum length for a C2 fragment defined operationally by reactivity with 12B2 antibody.

B S1 and S3 alleles, by virtue of limited geometries (modified from Chattopadhyay et al, 2005), may have access to distinct subsets of the full range of biological activities of WT PrP, which has access to interchangeable component 1, 2 and 3 geometries.

C Sequences of synthetic PrP alleles differing in the octarepeat region of mouse PrP. Conserved histidines in octarepeats 2–5 are coloured blue and missense mutations are shown in red. Overproduction of C2 fragment is indicated by ++ signs to the right of the panel.
Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation. (2015)
Figure 2

Western blot analysis of lysates from cells transiently transfected with plasmids encoding PrP mutants

A Lysates from RK13 cells transfected with WT, S1, S3 and S3.F88W PrP were probed with antibody Sha31, 9A2 and 12B2 after digestion with PNGaseF.

B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).

D Lysates of N2a, HEK, SMB-PS and SH-SY5Y cells not transfected (−) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31.

E Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP, S1 PrP and S3 PrP. Lysates were PNGaseF-digested before Western blot using the Sha31 antibody. F1: A114F, F2: G118F, F3: G122F, F4: G126F

Source data are available online for this figure.
Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation. (2015)
Figure 3

PrP expression in transgenic mice

A Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls.

B The ratio of C2 to full-length PrP in TgPrP(S3.F88W)-35 and WT mice (left panel, n = 7, P = 1.46E-06) and in RK13 cells expressing WT and S3 PrP (right panel, n = 5, P = 4.01E-05) is shown. Values are presented as mean ± SEM. Unpaired, two-tailed t-test, ****P < 0.0001.

C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.

Source data are available online for this figure.
Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation. (2015)
Figure 4

Variable rescue of the demyelinating polyneuropathy of Prnp0/0 mice by the S3 PrP allele

A Sections of sciatic nerve from TgPrP(S3.F88W)-14 female mice (all 610 days old) and TgPrP(S3.F88W)-35 female mice (614, 570 and 614 days old, respectively) were analysed using toluidine blue staining (upper row), with the third panels for each line showing a hypermyelinated phenotype. Scale bar in left panel represents 50 μm. The lower rows show EM analyses of mice of the indicated genotypes. Scale bar is 10 μm for panels 1–4, with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3.F88W PrP (scale bar, 5 μm).

B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.

C Quantification of fibre morphology in aged animals (492–608 days old), representing the percentage of the nerve occupied by fibres versus the total cross-sectional area of the nerve. The S1 and S3.F88W alleles rescue Prnp0/0 nerves to a similar percentage as the WT allele, but the variance and P-value is greater for the two Tg lines expressing the TgPrP(S3.F88W) allele. Unpaired, two-tailed t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Sample sizes and exact P-values compared with Prnp0/0 (n = 9) were as follows: TgPrP(WT) n = 6, P = 3.63E-05; Prnp0/0, n = 9; TgPrP(S1)-17 n = 5, P = 3.54E-05; TgPrP(S1)-19 n = 3, P = 6.35E-06; TgPrP(S3.F88W)-14 n = 3, P = 3.70E-03; and TgPrP(S3.F88W)-35 n = 7, P = 7.18E-03.

Source data are available online for this figure.
Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation. (2015)
Figure 6

Pathology of RML-infected mice

A-H The pattern of PrPSc deposition (A–D) and GFAP immunostaining (E–H) is presented.

I-P A higher power view of H&E stained hippocampus (I–L) and thalamus (M–P) to reveal vacuolation is presented.

Data information: Animals of the same genotype are shown within a column, and incubation times of the presented animals with the RML prion isolate are shown (days). Mice expressing PrP S1 and S3 alleles (central columns) exhibit attenuated PrPSc deposition, gliosis and spongy change. Pathological features of the RML isolate are not altered upon sequential passage through TgPrP(S3.88W)-35 mice (see schematic at bottom of the diagram indicating sequential passages and finally, passage back into WT mice, compare the data in first and fourth columns).
Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation. (2015)
Figure 7

Biochemical properties of PrPSc assessed by guanidine denaturation and CDI

A Spot counts in the scrapie cell assay using WT, TgPrP(S1)-17 and TgPrP(S3.F88W)-14 brain homogenates (0.1% or 0.01%) from mice infected with RML are shown in the upper panel (WT vs S1 P = 2.09E-05 and vs S3 P = 4.62E-08) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel (WT vs S1 P = 1.04E-13 and vs S3 P = 3.39E-20; two-tailed t-test, n = 3).

B A comparison of incubation time vs PrPC:PrPSc ratio for mice expressing different levels of WT PrP (WT, TgPrP(WT), tga20), S1 PrP (TgPrP(S1)-17, TgPrP(S1)-19, TgPrP(S1)-39) and S3 PrP (TgPrP(S3.F88W)-14, TgPrP(S3.F88W)-35).

C Formation and biological activities of PrP species. Synthetic and proteolytic pathways are shown from left to right, while pathways for pathologic signalling from PrPSc through PrPC and PrPC-derived fragments are shown from right to left (dashed arrows). Alternative pathways to remove N-terminal sequences (yellow) from the protease-resistant C-terminal Core (red) in infected cells are shown, while N2 is removed from a protease-sensitive C-terminal core (blue) in healthy cells. Grey arrow indicates that formation of PrPSc C2 from protease-sensitive C2 is not particularly efficient as mice with high levels of C2 are not more efficient in synthesizing PrPSc. Heavy dashed arrow through protease-sensitive C2 indicates that this species may be adept at pathogenic signalling as mice with lower PrPSc burden than WT mice succumb to disease earlier. For simplicity, physiological signalling pathways through PrPC species are not shown.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf1

(a–d) ELISA for IL-1β (a–c), IL-18 (d) and IL-6 (e) in supernatants of resting or LPS-primed bone marrow-derived macrophages (BMMs) stimulated with palmitate conjugated to BSA (PA-BSA) or BSA control. (e,f) ELISA for IL-1β (e) and IL-18 (f) in supernatants. Resting or LPS-primed BMMs generated from wild-type (WT), Nlrp3−/−, Pycard−/− or Nlrc4−/− mice were stimulated with PA-BSA. (g) IL-1β ELISA of supernatants from LPS-primed BMMs stimulated with PA-BSA in the absence or presence of the pan-caspase inhibitor zVAD or caspase-1 inhibitor zYVAD. (h) ELISA for IL-1β in supernatants. Resting or LPS-primed BMMs generated from WT or Casp1−/− mice were stimulated with PA-BSA, BSA control, ATP or nigericin. (i) ELISA for TNF in supernatants. Resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− or Casp1−/− mice were stimulated with PA-BSA as indicated. Values are expressed as mean ± s.d., and the results are representative of three independent experiments.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf2

Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf3

(a) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity. (b,c) ELISA for IL-1β (b) and immunoblotting for caspase-1 p10 and IL-1β p17 (c) on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC. (d) ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In a, one of two independent experiments is shown. In each of b–d, the results are representative of three independent experiments.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf4

(a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h. (b–e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (b) ROS production determined using the fluoroprobe DHR. (c,d) IL-1β (c) and IL-6 (d) in supernatants, measured by ELISA. (e) Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules. (f) Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. (g) ELISA for IL-1β in supernatants of cells transfected as in f and stimulated with PA-BSA (0.5 mM) for 24 h. In each of a, b and f, one of two independent experiments is shown. In each of c–e and g, the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P 0.05 versus controls.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf5

(a) Immunoblotting for AMPK activation (Thr172 p-AMPK-α) and LC3B (top), and densitometric analysis to quantify ratio of LC3B-II to actin (bottom). BMMs were pretreated with LPS (200 ng/ml) for 3 h, followed by PA-BSA (0.5 mM) treatment for 24 h in the absence or presence of chloroquine (50 μM). Med, medium control. (b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right). Cells were fixed and examined by transmission electron microscopy for autophagosomes (c). Quantitation (c, right) is based on counting autophagosomes in ten cells per treatment. (d) Immunoblotting (left) and densitometric analysis to quantify ratio of LC3B-II to actin and ULK1 species (right) in treated BMMs. (e) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator. In each of a and e, one of three independent experiments is shown. In each of b and d, one of two independent experiments is shown. Values are expressed as mean ± s.d. *P 0.05 versus controls.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf6

(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b). (c) p-IRS1 (Ser307), determined by immunoblotting, in FL83B mouse liver cells treated with either IL-1β or TNF for 24 h. (d–f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation. (g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin. (h) p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf7

(a) ITT performed 2 h after IL-1β injection. Eight-week old male C57BL/6 mice were intraperitoneally injected with saline or mouse recombinant IL-1β (1 μg/kg). (b) ITT in WT mice on regular diet (RD) or HFD for 12 weeks, or Il1b−/− mice on HFD for 12 weeks. (c,d) ITT (c) and serum IL-1β (d, left) and TNF (d, right) determined by ELISA, 2 h after IL-1β injection. Recombinant mouse IL-1β was administered to WT and Tnfa−/− mice (n = 5 for each group). One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling (2011)
figf8

(a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD. (b–e) Glucose tolerance test (GTT) (b,d) and insulin tolerance test (ITT) (c,e) performed for WT, Nlrp3−/− (b,c) and Pycard−/− (d,e) mice on HFD for 12 weeks. (f) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks. (g,h) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (g), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice (h) on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine. (i,j) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3−/− mice (i) and Pycard−/− mice (j) on regular diet (RD) or HFD for 12 weeks. One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.
Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex (2019)
Figure 3

Figure 3. Identification of residues required for interaction between hCAP-G and hCAP-H.

(A) Zoomed-in view of site IV. Residues of hCAP-G and hCAP-H are shown in orange and green, respectively. The dashed red lines indicate hydrogen bonds.

(B) 3Q, 5Q, 3A, 5A, 2A and Δ506-515 mutants of hCAP-H. Motif IV (residues 461-503) contains amino-acid residues highly conserved among eukaryotic species (X, Xenopus laevis; Dr, Danio rerio; Cm, Cyanidioschyzon merolae; Sp, Schyzosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ec, Encephalitozoon cuniculi). To produce the IV-3Q, 5Q, 3A, 5A and 2A mutants, the conserved aromatic amino-acid residues (F463, F469, F473, F501 and Y503; labeled in dark blue) were substituted with glutamine (Q) or alanine (A) residues. The secondary structural element of hCAP-H is drawn below the sequence alignments.

(C) Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1-478, 554-900) and hCAP-H (residues 394-515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6) or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506-514 residues were deleted from 394-515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS-PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9).
Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex (2019)
Figure 4

Figure 4. CAP-H motif IV is required for a physical interaction with CAP-G.

(A) Expression of condensin I subunits in insect cells. The wild-type (WT) or IV-5Q mutant CAP-H subunit was co-expressed with the other four subunits (GST-SMC4, SMC2, CAP-D2 and CAP-G) in insect cells. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 (left panel) or against CAP-D2, -G, and -H (right panel).

(B) Purification of the WT and IV-5Q mutant condensin I complexes. Protein samples purified through glutathione-affinity chromatography were subjected to SDS-PAGE and analyzed by CBB staining (left panel) or immunoblotting with a mixture of antibodies as described above (middle and right panels).

(C) Add-back assay using the WT and mutant condensin I complexes. Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes (from top to bottom; WT, IV-5Q, ∆G, ∆G[IV-5Q]). The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes. The samples were fixed and labeled with an antibody against mSMC4 (red). DNA was counterstained with DAPI (blue). The data from a single representative experiment out of two repeats are shown. In the experiment shown here, multiple images were collected for condensin-depleted extracts supplemented with the WT (n = 17), IV-5Q (n = 22), ∆G (n = 20) and ∆G(IV-5Q) (n = 25) complexes. The scale bar represents 10 μm.
Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex (2019)
Figure 5

Figure 5. DNA-binding surfaces conserved between hCAP-G and YCG1.

(A) Purification of hCAP-G and hCAP-G-H subcomplexes: wild type (WT), CAP-G K60D/R848E double mutant (K60D/R848E) and CAP-G R168E mutant (R168E). Purified protein samples were subjected to SDS-PAGE and analyzed by CBB staining.

(B) Double-stranded DNA (dsDNA) and single-stranded DNA binding assay of the hCAP-G. 30-bp dsDNA was incubated with no protein (lanes 1) or increasing amounts of WT hCAP-G (WT; lanes 2-4). 30-mer ssDNA was incubated with no protein (lanes 5) or increasing amounts of WT hCAP-G (WT; lanes 6-8).

(C) The dsDNA binding assay for the hCAP-G-H subcomplexes. 30-bp dsDNA was incubated with no protein (lanes 1, 5 and 9), increasing amounts of WT hCAP-G-H subcomplex (WT; lanes 2-4), CAP-G K60D/R848E double mutant (K60D/R848E; lanes 6-8) or CAP-G R168E mutant (R168E; lanes 10-12).

(D) The ssDNA binding assay for the hCAP-G-H subcomplexes used in panel (C).

(E) The molecular surface of hCAP-G in complex with hCAP-H. The structural model of hCAP-G is shown in white. hCAP-H is shown as a green ribbon model. Identical and homologous residues between hCAP-G and YCG1 are shown in blue and cyan, respectively.

(F) Zoomed-in view of the HEPES-binding site. R168 of hCAP-H interacts with HEPES.

Expanded View Figure legends
The NFIB-ERO1A axis promotes breast cancer metastatic colonization of
disseminated tumour cells (2021)
Figure 1

Figure 1. Transposon mutagenesis screen identifies Nfib as a candidate metastatic inducer in breast cancer.

A Screen design: generation of mammary cancer cell lines LB-mHR1 (HR1) from PIK3CAH1047R mutant transgenic animals and transfection with the piggyBac (PB) system (HR1 Ctrl and HR1.PB cells) (Ivics et al, 2009). PB transposon (ATP1-Puro) and transposase (pFS250.PBase) plasmid design. PB-3'/5', piggyBac inverted terminal repeats; CβASA, Carp β-actin splice acceptor; En2SA, Engrailed-2 exon-2 splice acceptor; SD, Foxf2 exon-1 splice donor; pA, bidirectional SV40 polyadenylation signal; PGK, mouse phosphoglycerate kinase 1 promoter; Puro, puromycin resistance; CAG, cytomegalovirus enhancer and chicken beta-actin promoter; Tet-ON, tetracycline-responsive element-tight promoter; PBase, PB transposase; UbC, Ubiquitin C promoter; rTTA3, reverse tetracycline transactivator 3; IRES, internal ribosomal entry site; Neo, Neomycin/G418 selection marker. The cell pools were treated with doxycycline (1 μg/ml) for 96 h in vitro.

B In vivo screen design: generation of lung metastatic mammary cancer cell lines (LM) from HR1.PB cell lines after orthotopic injections of HR1.PB or HR1.Ctrl.

C Bar graph showing the percentage of unique insertions in the top 7 genes normalized to the total number of insertions in a given sample. Genes are annotated with a gene symbol when available otherwise with UCSC ID transcript names. Data are from all the sequenced samples (tumour samples: 16 primary tumours; metastatic samples: 3 lung micro-metastases, 6 lung macro-metastases and 9 LM cell lines). Means ± s.d., two-tailed Mann-Whitney U-test, n.s. = not significant.

D Bar graphs showing the percentage of unique insertions in Nfib and Foxp1 in tumours, LM1, LM8, LM9, and other LM cell lines normalized to the total number of insertions in a given sample. Dots represent individual samples (Tumours n = 16, LM1/8/9 n = 9, Other LM n = 9), means ± s.d., two-tailed Mann-Whitney U-test, n.s. = not significant.

E Bar graphs representing mean Nfib mRNA expression normalized to HR1 cells. n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test, FC= fold change.

F Bar graphs representing mean Foxp1 mRNA expression normalized to HR1 cells. n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test, FC= fold change.

G Immunoblot analysis of LM and HR1 cell lines showing NFIB and FOXP1 protein levels. ERK2 served as a loading control.