1. Volume 123, Issue 6, Pages 1980-1991 (December 2002)
Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis 
Pierre H. Vachon, Charlène Harnois, Amélie Grenier, Geneviève Dufour, Véronique Bouchard, Jiahuai Han, Jacques Landry, Jean– Fraçois Beaulieu, Anne Vézina, Anders Bondo Dydensborg, Rémy Gauthier, André Côté, Jean–Fraçois Drolet, Fraçois Lareau 
Gastroenterology 
Volume 123, Issue 6, Pages (December 2002)
DOI: /gast
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2. Fig. 1
The time-course activation kinetics of p38 correlate with enterocytic anoikis. (A) Representative DNA laddering assays from undifferentiated (−2PC) and differentiated (30PC) Caco-2/15 cell cultures maintained for 0–12 hours in suspension, in serum-free medium; L, 100-bp DNA size markers. (B) Representative Western blot analyses of p38 activation from HIEC (undifferentiated) cells adhering (control) or in suspension (anoikis) for 0–24 hours in serum-free medium. Specific antibodies for the detection of the activated doubly-phosphorylated form of p38 (ppp38) and p38 protein (total p38) were used. (C) Same as in B, except that 30PC (differentiated) Caco-2/15 cells were analyzed.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Kinetics of p38 activation during enterocytic anoikis. (A, B) Relative activation levels of p38 in (A) HIEC cells and (B) 30PC Caco-2/15 cells maintained for 0–24 hours in serum-free medium (control; ■), or with the addition of 1 μmol/L cytochalasin D (○), with the addition of 30 μmol/L Ly (□), or kept in suspension (●). After Western blot analyses with specific antibodies for the detection of the activated doubly phosphorylated form of p38 (ppp38) and p38 protein, immunoreactive bands were semiquantified and the relative activation levels of p38 were established with the ratios ppp38/p38. ■, control; ○, CD; ●, susp.; □, Ly.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
The Fak/PI3-K/Akt pathway and p38 isoforms in the survival and suppression of enterocytic anoikis. Schematic drawing of an undifferentiated enterocyte (left) and its differentiated counterpart (right), showing how integrin-mediated cell adhesion can stimulate the Fak/PI3-K/Akt pathway to promote cell survival and suppression of anoikis. The drawing also summarizes the main results of the present study concerning the down-regulation of the Fak/PI3-K/Akt pathway in anoikis, the p38 isoforms expressed distinctively in undifferentiated and differentiated enterocytes, and the p38 isoforms selectively implicated in enterocytic anoikis according to the state of differentiation. Question marks indicate the possible role of Akt in inhibiting the activation of specific p38 isoforms (see Discussion section).
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Time-course down-regulation of Fak and Akt during enterocytic anoikis. (A) Representative Western blot analyses from Fak and Akt immunoprecipitates of HIEC cells maintained in suspension for 0–24 hours in serum-free medium. Specific antibodies for the activated phosphorylated forms of Fak (pp125Fak), Akt (pp57Akt), and respective total protein forms (p125Fak, p57Akt), were used. (B) Same as in A, except that immunoreactive bands were semiquantified and the relative activation levels of Fak and Akt were established with the ratios phospho-protein/total protein (pp125Fak/p125Fak, ○; pp57Akt/p57Akt, ●).
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Anoikis leads to a down-regulation of Fak and Akt in enterocytes. (A, B) HIEC (undifferentiated) and 30PC Caco-2/15 (differentiated) cells maintained for 24 hours in serum-free medium (control) with the addition of 1 μmol/L cytochalasin D (●), with the addition of 50 μg/mL nonimmune mouse immunoglobulin G (□), with the addition of 50 μg/mL of the monoclonal antibody P4C10 (■), or kept in suspension (●). Cultures were harvested and processed for Western blot analyses of Fak and Akt activation by using specific antibodies for the detection of the activated phosphorylated forms of Fak (pp125Fak), Akt (pp57Akt), and respective total protein forms (p125Fak, p57Akt). Immunoreactive bands were semiquantified and the relative activation levels of Fak and Akt were established with the ratios (A) pp125Fak/p125Fak and (B) pp57Akt/p57Akt, which in turn were compared with those of control cultures, ×100 (expressed as % of control). *Statistically significant (0.001 ≤ P ≤ 0.01) differences between treatments and controls.
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7. Fig. 6
Intestinal epithelial cells express distinct profiles of p38 isoforms according to their state of differentiation. (A) Representative reverse-transcription polymerase chain reaction analyses of the expression of p38 isoform mRNAs in HIEC (undifferentiated; U; lane 1) and 30PC Caco-2/15 (differentiated; D; lane 2) cells. Total RNA was extracted, reversed-transcribed, and then 20 cycles of amplification were performed by using primers specific for human p38α (199-bp fragment), p38β (310-bp fragment), p38γ (400-bp fragment), or p38δ (494-bp fragment). S14 mRNA expression also was analyzed for normalization purposes. (B) Same as in A, except that analyses were performed also in −2PC Caco-2/15 (undifferentiated; ●) and pure culture of differentiated enterocytes (differentiated; ●) cells, in addition to HIEC (undifferentiated; □) and 30PC Caco-2/15 (differentiated; ■) cells. Amplified bands were semiquantified and the relative expression levels of p38α, β, γ, and δ mRNAs were established with the ratios p38 isoform/S14.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
CAD-associated DNA laddering and caspase-mediated cleavage of PARP are attenuated when p38(α/β) kinase activity is inhibited in undifferentiated, but not differentiated, enterocytes. (A) Representative DNA laddering assays from HIEC (undifferentiated) and 30PC Caco-2/15 (differentiated) cell cultures maintained for 24 hours in serum-free medium (control; lanes 1, 7) with the addition of 1 μmol/L cytochalasin D (lanes 3, 4, 9, 10), with the addition of 20 μmol/L SB (lanes 2, 4, 6, 8, 10, 12), or kept in suspension (lanes 5, 6, 11, 12). L, 100-bp DNA size markers. (B) Representative Western blot analysis of PARP from HIEC cells maintained for 24 hours in serum-free medium and adhering (control; lane 1) or kept in suspension (lane 2), with or without 20 μmol/L SB (lane 3). A specific antibody for the simultaneous detection of intact PARP (p116PARP) and its caspase-generated 89-kilodalton proteolytic fragment (p89) was used.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
The inhibition of p38(α/β) kinase activity attenuates anoikis in undifferentiated, but not in differentiated, enterocytes. (A) HIEC and (B) Caco-2/15 30PC cultures were maintained for 24 hours in serum-free medium (control) with the addition of 1 μmol/L cytochalasin D, 50 μg/mL nonimmune mouse immunoglobulin G or 50 μg/mL of the monoclonal antibody P4C10, with (or without) 20 μmol/L SB and/or 20 μmol/L PD ISEL was then performed to establish the apoptotic indices, expressed as the percentage of apoptotic cells over the total number of cells counted. *, ** Statistically significant (0.001 ≤ P ≤ 0.01) differences between anoikis-inducing treatments and controls, as well as between treatments and their counterparts with SB and/or PD98059, respectively. (C) HIEC (□) and −2PC Caco-2/15 (■) cells were transfected transiently with the empty vector pCMV-Neo or a vector containing a cDNA coding for FRNK (pCMV-p45FRNK), or sham-transfected (control). Cells were thereafter kept in serum-free medium for 24 hours with (or without) 20 μmol/L SB ISEL was then performed to establish the apoptotic indices, which in turn were compared with those of control cultures, ×100 (expressed as % of control). *, **Statistically significant (0.001 ≤ P ≤ 0.01) differences between treatments and controls, as well as between treatments and their counterparts with SB202190, respectively.
Gastroenterology  , DOI: ( /gast )
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10. Fig. 9
p38β is involved specifically in anoikis of undifferentiated enterocytes. (A) HIEC (undifferentiated; U; lanes 1–3) and 30PC Caco-2/15 (differentiated; D; lanes 4–6) cells were maintained in serum-free medium (control; lanes 1 and 4) with the addition of 1 μmol/L cytochalasin D (lanes 2 and 5), or kept in suspension (lanes 3 and 6). After 4 hours, cells were lysed and p38α was immunoprecipitated (IP) with a specific antibody. Total proteins from immunoprecipitates were probed (Western blot [WB]) with specific antibodies for the detection of the activated doubly phosphorylated form of p38 (ppp38) and p38α protein (p38α). (B) Same as in A, except that p38β was analyzed after its immunoprecipitation with a specific antibody.
Gastroenterology  , DOI: ( /gast )
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11. Fig. 10
Differentiation state-selective roles of p38 isoforms in intestinal epithelial cell survival and anoikis. Caco-2/15 cells were stably transfected with either pcDNA3 (control; lanes 1 and 5), a dominant-negative activation-dead p38β mutant (p38βAGF-Flag; lanes 2 and 6), a dominant-negative kinase-dead p38γ mutant (p38γKM-Flag; lanes 3 and 7) or a dominant-negative kinase-dead p38δ mutant (p38δKM-Flag; lanes 4 and 8). Upper panels: representative Western blot analyses for the expression of the Flag epitope to confirm expression of cDNA constructs in stably transfected cells in their undifferentiated (lanes 1–4) and differentiated (lanes 5–8) states. Lower panels: representative DNA laddering assays of stably transfected cells that were kept in suspension for 12 hours while being still undifferentiated (lanes 1–4) or after allowing them to reach their differentiated (lanes 5–8) state; L, 100-bp DNA size markers.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions