EMBO/soda-nel huggingface dataset:
CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. (2014)
Figure 5.

Dynamic interaction of the CIP2A, PP2A, and mTORC1 complex regulates mTORC1 activity. (A and B) In vitro translated full-length CIP2A was incubated with purified human PP2A complex and HA-Raptor (A) or HA-S6K1 (B) purified from transiently transfected MCF7 cells. Protein complexes were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting as indicated. Nonspecific IgG served as a control. Note that human reticulocyte (RC) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac. (C) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right. (D) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control (Ctr) or CIP2A shRNA lentiviruses (Fig. 3 A) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right. (E) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control (C) or PPP2R1A (R1A) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies. Immunoblots of corresponding cell lysates are shown on the left. (F) HA-S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses (Fig. 3 A), transiently transfected with HA-RPS6K1 cDNA and starved for amino acids for 20 min when indicated, were analyzed by immunoblotting. Immunoblots of corresponding cell lysates are shown on the right. (G) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full-length CIP2A. Error bars are SDs for four (D) independent experiments or a representative triplicate experiment (C and G). (H) Representative confocal images of untreated and amino acid–starved (4 h) MCF7 cells stained for endogenous CIP2A and raptor. Black lines indicate that intervening lanes have been spliced out. −AA, amino acid starvation; IP, immunoprecipitation; a.u., arbitrary unit. Bar, 10 µm.
CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. (2014)
Figure 6.

CIP2A and c-Myc are degraded in an autophagy-dependent manner. (A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0–24 h. (B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments. (C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control. (D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0–5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA. (E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm. (F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control. (G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (<100 kD), medium (>150 kD), and long (100–130 kD) exposures are shown. (H) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions (see G) and analyzed by immunoblotting for CIP2A and monoubiquitin. (I) Lysates from MCF7 cells transfected with the indicated ubiquitin-HA constructs were subjected to anti-CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA. (J) Lysates from MCF7 cells transfected with the CIP2A-FLAG construct (Junttila et al., 2007) and the indicated ubiquitin-HA constructs were subjected to anti-FLAG immunopurification in denaturing conditions with 0.1% SDS followed by immunoblotting with the indicated antibodies. Black lines indicate that intervening lanes have been spliced out. Ub, ubiquitin; WT, wild type.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 1

Structure‐based analysis of Gli1/DNA complex

Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres.
Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.
Effect of Gli1ZF mutants on Gli1‐dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS‐Luc (GliBS, Gli binding site), pRL‐TK Renilla (normalization control), Flag‐Gli1 WT or the indicated Flag‐Gli1 mutants. Data show the mean ± SD of three independent experiments. *P 0.01; **P 0.05 versus Gli1 WT. Western blot analysis of Flag‐Gli1 WT or the indicated Flag‐Gli1 mutant expression levels (bottom panel).
Specific H‐bond interactions between K350 of Gli1ZF and Gli1 binding site consensus, as predicted by MD.
Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′–TTGCCTACCTGGGTGGTCTCTCCACTT–3′) or mutated GliBS sequence used as control (5′–TTGCCTACCTCCCACTTCTCTCCACTT–3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242–424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.

Source data are available online for this figure.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 2

GlaB inhibits Hh signaling by impairing Gli1 function

Inhibition of Gli1‐induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only.
GlaB chemical structure with numbering scheme for NMR analysis.
Inhibition of Gli1‐induced transcription in transfected Smo−/− MEF cells. Smo−/− MEF cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control.
The graphs show the Hh target gene expression levels in Ptch1−/− MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real‐time PCR (qRT–PCR) normalized to endogenous control (β2‐microglobulin and HPRT). Pfkfb3 gene was used as a negative control.
Representative model of Hh signaling hyperactivation: in Ptch1−/− MEFs, constitutive activation of Smo and consequently of Gli1; in SuFu−/− MEFs, release of Gli1 suppression.
SuFu−/− MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1 mRNA levels were determined by qRT–PCR normalized to β2‐microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control.
Promoter occupancy of Gli1 is prevented by GlaB treatment. MEF WT cells were transfected with Flag‐tagged Gli1 or empty vectors, and chromatin immunoprecipitation (ChIP) was carried out. qRT–PCR was performed using primers encompassing the Gli‐BS of mouse Ptch1 promoter (right, schematic representation). Results are indicated as fold difference, relative to empty (pcDNA3) control.
Ptch1 mRNA expression levels (left panel) were determined by qRT–PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non‐specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2 mRNA expression levels determined by qRT–PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression.

Data information: All data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 3

Analysis of Gli1/GlaB interaction

The predicted binding mode of GlaB (green sticks) to Gli1ZF (blue cartoons). Residues highlighted by the single‐point mutation study to impact on Gli1 binding to DNA are shown as blue sticks; K340 and K350 of the GlaB binding site are colored magenta. GlaB protons highlighted by the NMR study are shown as orange spheres.
Detail of GlaB binding to Gli1ZF‐WT, Gli1ZF‐K340A, GLI1ZF‐K350A, GLI1ZF‐K340A/K350A mutants and respective theoretical affinity values. Gli1 is shown as blue transparent surface; K340 and K350 are colored magenta. GlaB is shown as green sticks.
Inhibition of Gli1/DNA binding by GlaB. EMSA using recombinant GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A in the presence of different concentrations of GlaB or with DMSO only. The shifted complex is competed with a 50× excess of cold probe. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding in absence of GlaB. *P 0.05 versus DMSO; **P 0.05 K340A + GlaB versus Gli1 WT + GlaB.
HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty vector) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB. Treatment time was 24 h, and control cells were treated with DMSO only. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO; **P 0.05 K340A versus Gli1 WT.

Source data are available online for this figure.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 4

GlaB inhibits Gli1‐dependent growth of cerebellum‐derived normal progenitors

A qRT–PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.c. injections of GlaB.
B Representative EGL HE staining of 6‐day‐old mouse cerebellum after s.c. injections of GlaB (upper panel); immunohistochemistry shows Gli1 (middle panel) and Ki67 stainings (lower panel). In all images, the scale bar represents 50 μm.
C The graph shows the percentage of EGL thickness reduction in GlaB‐treated mouse cerebella in comparison with vehicle mouse cerebella. Data show the mean ± SD of cerebella (n = 10) for each treatment. *P 0.05 versus Ctr.
D BrdU assay in GCPs. Cerebellar granule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouse Sonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh.
E, F qRT–PCR and Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mouse cerebella after s.c. injections of GlaB. In all qRT–PCR experiments, the results were normalized to endogenous control (β2‐microglobulin and HPRT). *P 0.05 versus Ctr.

Source data are available online for this figure.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 5

Inhibition of Gli‐dependent MB tumor cell growth

A, B Ex vivo cell cultures from Ptch1+/− mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells. Gli1 mRNA expression levels were determined by qRT–PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT).
C–F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/− mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right). (D, E) MB‐SCs isolated from Ptch1+/− mice MBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT–PCR and Western blot analysis show Hh, proliferation and stemness target mRNA and protein expression levels. For qRT–PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.
G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/− mice MBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.

Data information: In all experiments, data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.Source data are available online for this figure.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 6

Ptch1+/− MB allografts

A Change of tumor volume during GlaB or vehicle treatment period (18 days).
B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05.
C Representative Masson's trichrome staining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichrome staining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings.
D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05.
E, F qRT–PCR and Western blot analysis show Hh and proliferation target mRNA and protein expression levels. For qRT–PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 7

Orthotopic medulloblastoma xenograft models

Representative images (low and high magnification) of HE (upper panel) and Ki67 immunohistochemical staining (lower panel) of a human Daoy MB cell‐derived orthotopic tumor in NOD/SCID mice cerebella after i.p. injections of GlaB. Scale bars represent 500 or 200 μm for HE staining, and 150 μm or 50 μm for Ki67 staining.
Representative tumor average volumes at the end of treatment. Error bars indicate SD. *P 0.05.
Quantification of Ki67 staining from immunohistochemistry shown in (A). Error bars indicate SD. *P 0.05.
Gli1/DNA interaction is a druggable target for Hedgehog‐dependent tumors (2014)
Figure 8

Inhibition of Gli‐dependent BCC tumor cell growth

A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.
C Gli1 mRNA expression levels were determined by qRT–PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.
D–H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (D) Change of tumor volume during GlaB or vehicle treatment period (18 days). (E) Representative flank allografts average volumes (upper panel). (F) HE and immunohistochemical staining of Gli1, Ki67 and TUNEL of allograft tumor samples. Scale bars represent 50 μm for HE, 25 μm for Gli1, 25 μm for Ki67 and 30 μm for TUNEL stainings. (G) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (F). (H) qRT–PCR of Hh and proliferation target mRNA expression levels. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Shown is the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 1

Figure 1 - Skin fibroblasts derived from HGPS patients display deficiency in ciliogenesis.

A Normal human fibroblasts and HGPS fibroblasts were serum-starved for 48 h and stained for progerin (red), acetylated tubulin (green) and DNA (blue). The representative images show that the cell with high progerin (inset 2), but not low progerin (inset 1), displays defective cilia formation. Scale bars, 20 μm or 2 μm (in the magnified images).

B The progerin fluorescence intensity of the cells was measured by the Zeiss ZEN2 software (n ≥ 148). The dashed line indicates the background level of progerin. A.U., arbitrary units.

C The cells were grown in the growth medium and stained for progerin (red) and F-actin with phalloidin (green). The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3.5 x 106 (A.U.), respectively. The cell spreading area was measured according to the phalloidin staining (n ≥ 30). Scale bars, 50 μm.

D The cells were serum-starved for 32 h to allow cilia formation and then treated with (+) or without (-) 250 nM cytochalasin D (Cyto D) for another 16 h before fixation. The cells were stained for progerin (red), acetylated tubulin (green), F-actin (white), and DNA (blue). The numbers of actin filaments within 60 μm2 around the cilia (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (n ≥ 55). Scale bars, 20 μm or 2 μm (in the magnified insets).

E,F The cells were subjected to immunofluorescence stain as in (D). The percentage of the cells with cilia in the total counted cells (E, n ≥ 50) and the cilia length (F, n ≥ 50) were measured.

Data information: In B, C, D and F, values (means ± s.e.m.) are from three independent experiments. In E, values (means ± s.d.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t-test.
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 2

Figure 2 - Lmna null mice display defective primary cilia in the skeletal muscle, kidney, uterus and ovary.

A The skeletal (tabialis anterior) muscles from control (Lmna+/+ and Lmna+/-) and Lmna-/- mice were stained for acetylated tubulin (green) and nucleus (blue). The representative images with low and high magnification are shown. The insets are enlarged images for cilia.

B The kidneys from control and Lmna-/- mice were stained for acetylated tubulin (green) and AQ1 (a marker for the proximal tubules, red). The insets show the cilia of proximal renal tubule cells.

C The uterus from control and Lmna-/- mice was stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia at the stroma of the uterus.

D The ovary from control and Lmna-/- mice were stained for acetylated tubulin (green) and keratin 8 (red). The insets show the cilia of the granulosa cells in the follicles of the ovary.

E, F The cilia length (E, n=150-180) and cilia positive cells (F, n ≥ 1197) in each organ from 4-week-old Lmna-/- mice (n=3) and their littermate controls (n=3) were measured.

Data information: Scale bars, 200 μm (A-D, upper panels); 10 μm (A-D, lower panels); 1 μm (A-D, insets). In E, values (means ± s.e.m.) are from three independent experiments. In F, values (means ± s.d.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t-test.
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 3

Figure 3 - Depletion of lamin A/C expression causes impaired ciliogenesis in RPE cells.

A RPE cells were infected with lentiviruses encoding shRNAs specific to lamin A/C (shLamin A/C) or luciferase (shLuc). Exogenous lamin A was re-expressed in the lamin A/C-depleted cells (shLamin A/C-Lamin A). An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated.

B The cells were serum-starved for 48 h and stained for acetylated tubulin (green), γ-tubulin (red) and DNA (blue). The depletion of lamin A/C (shLamin A/C) led to impaired ciliogenesis, as manifested by no cilia (inset 1) or short cilia (inset 2). Scale bars, 20 μm or 2 μm (insets).

C The percentage of the cells with cilia in the total counted cells (n ≥ 626) was measured.

D The length of cilia was measured (n ≥ 87 in each group).

E Cells were stained for acetylated tubulin (green) and γ-tubulin (red) after various intervals of serum starvation. The percentage of cilia-positive cells at each time point (n ≥ 372) was measured.

Data information: In C and E, values (means ± s.d.) are from three independent experiments. In D, values (means ± s.e.m.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t-test.
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 4

Figure 4 - Depletion of lamin A/C increases the formation of F-actin, which has an adverse effect on ciliogenesis in RPE cells.

A-C The RPE cells as described in Fig 3 were stained for paxillin (as a marker for focal adhesion; green), F-actin (red) and DNA (blue). The representative images are shown (A). Scale bars, 20 μm or 5 μm (insets). The cell spreading area of the cells (B, n ≥ 108) and the number of focal adhesions per cell (C, n ≥ 30) were measured.

D-G The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D (250 nM) for another 16 h. The cells were stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (D). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of actin filaments within 60 μm2 around the basal body (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (E, n ≥ 132). The percentage of the cells with cilia in the total counted cells (F, n ≥ 357) and the length of cilia (G, n ≥ 126) were measured.

Data information: In B, C, E and F, values (means ± s.d.) are from three independent experiments. In G, values (means ± s.e.m.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t-test. For clarity, not all significances are indicated.