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1.
Figure 10

Figure 10. Induction of p21cip1/Waf1 messenger RNA and protein in Klf4 null lung versus normal lung at birth.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

The content of p21cip1/Waf1 mRNA increased 38-fold in Klf4 knockout lung between day 19 of gestation and 6 h after birth, but only 5-fold in normal lung.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
2.
Figure 4

Figure 4. Klf4 Immunohistochemistry.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

Klf4 protein is detected within nuclei of interstitial cells and airway epithelial cells of perinatal lung in the left panel. Signal is absent with omission of primary antibody as a negative control in the middle panel. Signal is present in epithelial cell nuclei of adult colon as a positive control in the right panel.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
3.
Figure 11

Figure 11. Immunohistochemisry for p21cip1/Waf1 protein.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

An intense nuclear immunohistochemical signal (brown color) for p21cip1/Waf1 protein is present in numerous interstitial (IC) cells (top left panel) and airway epithelial (A) cells (top right panel) of Klf4 null lung at birth. Signal is sparse in normal lung (bottom left panel) and absent with omission of primary antibody as the negative control (bottom right panel).

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
4.
Figure 5

Figure 5. Regulation of fibroblast Klf4 messenger RNA expression by oxygen.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

Exposure of mouse lung fibroblasts (MLg cells) to 95% oxygen induces Klf4 mRNA by 2.1 fold after 12 h (n = 4, p<0.05) and 2.8-fold by 48 h (n = 4, p<0.05). Klf4 mRNA induction in MLg cells exposed to 95% oxygen is blocked by pre-treating the cells with actinomycin D (95%+Actin D), but not cycloheximide (95%+CHX). Asterisks denote p<0.05.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
5.
Figure 14

Figure 14. Birth shock hypothesis.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

We propose that the changing alveolar oxygen at birth changes the local redox state (birth shock) and thereby acts as a physiologic signal to induce Klf4 mRNA expression, which then regulates proliferation and apoptosis in fibroblasts and airway epithelial cells, and connective tissue gene expression and myofibroblast differentiation at the tips of developing alveoli.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
6.
Figure 9

Figure 9. Nuclear localization of p53 protein.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

An intense immuno-histochemical signal (brown color) for p53 protein is present in numerous airway epithelial (A) and mesenchymal cells of newborn Klf4 null mouse lung (top left panel). Few cells in normal lung exhibit any signal (top right panel). Signal is present in macrophages of the hyperoxia-exposed GGTenu1 mouse lung (arrows) as a positive control [13] (bottom left panel). No signal is present with omission of primary antibody as a negative control (bottom right panel).

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
7.
Figure 6

Figure 6. Glutathione disulfide accumulation at birth.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

Glutathione disulfide (GSSG) was measured in the lung and the liver of mice just before birth (time 0) and then 2 h, 6 h and 12 h after birth in room air and at similar times in the lungs of mice born in 10% oxygen. Asterisks denote p<0.05 compared to value at time 0.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
8.
Figure 3

Figure 3. Confirmation of Klf4 mRNA Induction in the Lung at Birth.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

Klf4 mRNA induction was confirmed by Northern analysis (2.5 fold at 2 h and 3-fold at 6 h in 21% oxygen, n = 3, p<0.05). Klf4 mRNA induction at 2 h was attenuated by birth in 10% oxygen (1.7 fold, n = 3, p>0.05) and a shift of the induction response to the right. Asterisks denote significant increase above that of F21 lung in room air.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
9.
Figure 8

Figure 8. Proliferating cell nuclear antigen (PCNA) immunohistochemistry.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

An intense immunohistochemical signal (brown color) for PCNA protein is present in numerous mesenchymal and airway epithelial (A) cell nuclei from Klf4 null mouse lung (top left panel). Few cells of the normal lung exhibit any PCNA signal (top right panel). PCNA signal is present in proliferating epithelial cells of the colon as a positive control (bottom left panel). PCNA signal is absent with omission of primary antibody as negative control (bottom right panel).

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
10.
Figure 12

Figure 12. Lung cell apoptosis at birth.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

An intense nuclear immunohistochemical signal (brown color) for caspase-3 protein is present in numerous airway epithelial (A) and surrounding interstitial cells but absent from vascular (V) cells in Klf4 null mouse lung (top left panel). Few cells (arrow) of the normal lung exhibit signal (top right panel). Signal limited to airway epithelial (A) cells in hyperoxia-exposed GGTenu1 lung as a positive control [13] (bottom left panel). No signal with omission of primary antibody as the negative control (bottom right panel).

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
11.
Figure 2

Figure 2. Number of genes changing their level of expression at birth.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

In the lung birthed in room, the greatest number of gene changes (157) was at 2 h and transcriptional regulation, structure, apoptosis and antioxidant activity were overrepresented functional categories. Thereafter the numbers declined to 113, 99 and 57 at 6 h, 12 h and 24 h, respectively. In the liver birthed in room air, there were only 59 gene changes at 2 h and 100 at 6 hr. In the lung birthed into 10% oxygen, there were only 65 gene changes at 2 h and 80 at 6 h. No functional category of gene expression was overrepresented in either dataset.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
12.
Figure 13

Figure 13. Immunohistochemistry for smooth muscle actin in lung at birth.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

In the normal lung at 6 hours after birth, a specific smooth muscle actin (SMA) signal (green) is present in myofibroblasts at the saccular tips (tips) and vascular smooth muscle cells from a large blood vessel (V) in the left panel (green fluorescence) and the right panel (green despite merger with red autofluorescence). Nonspecific yellow signal (green merged with red autofluoresecence) is present in hematopoietic cells in large blood vessels. In the Klf4 null lung, however, the specific green SMA signal is only present in vascular smooth muscle cells from a large blood vessel (V). Cells in saccular tips in the left panel are yellow after merger with red autofluorescent signal in the right panel denoting non-specific signal likely originating from hematopoietic cells in small blood vessels within the lung interstitium as in large blood vessels.

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
13.
Figure 1

Figure 1. Transcriptional profile of gene expression in lung at birth in room air.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

Heirarchical cluster of the 157 genes that were up-regulated (red) or down-regulated (green) after birth (B) in room air at 2 h (B +2) and 6 h (B +6) compared to one day prior to birth on the last day of gestation (fetal day 21: F21). Four patterns are evident: transient up or down regulation and persistent up or down regulation. Arrow denotes Kruppel-like factor 4 (Klf4), a transcriptional regulator that exhibited the highest t-test (p<0.00001) and the greatest fold change at birth (5-fold).

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.
14.
Figure 7

Figure 7. Decrease in connective tissue gene expression in perinatal lung with Klf4 deficiency.. From: Transcription Factor Klf4, Induced in the Lung by Oxygen at Birth, Regulates Perinatal Fibroblast and Myofibroblast Differentiation.

(A) Cells from the Klf4 null lung were immortalized with SV40Tag as described in Methods. These Klf4 null fibroblasts (Fib-Klf4) express vimentin, the alpha 1 chain of Type 1 collagen (Col a1) and smooth muscle actin (SMA) in common with the mouse lung fibroblasts (MLg), but neither SP-C nor cytokeratin 8 which are found in mouse lung epithelial cells (MLE). The ribosomal marker 28S is shown as a loading control. (B) Klf4 cDNA was constitutively expressed in Fib-Klf4 cells to generate Fib+Klf4 cells as described in Methods. Compared to the empty vector control cells (C), these Klf4 expressing fibroblasts (Fib+Klf4) exhibit up-regulated expression of messenger RNA for p21cip1/Waf1 (2 fold), SMA (3.5 fold), the alpha 1 chain of Type 1 collagen, tenascin C and fibronectin (2–2.5 fold each) but not fibronectin receptor beta (Itgb1). The ribosomal marker 18S is shown as a loading control. These data represent two independent Northern blot experiments. (C) Comparative levels of expression for these Klf4 target genes in Klf4 null lung (null) relative to normal lung (Nor) at fetal day 19 of gestation and 6 h after birth in room air (n = 3 for each sample, asterisk denotes difference in level of expression in Klf4 null lung versus normal lung at p<0.05).

Jyh-Chang Jean, et al. PLoS One. 2013;8(1):e54806.

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