In vivo detection of δ-crystallin and CP49 by overexpressing L-Maf, MafB, and c-Maf, showing different inducing ability

Whole-mount immunostaining using δ-crystallin antibody was performed using stage 16 embryos electroporated with empty vector as negative control (,), wild-type L-Maf (,), c-Maf (,), and MafB (-) at stage 10. Embryo electroporated with empty vector showed no expression of δ-crystallin outside lens (). Ectopic δ-crystallin expression was detected in all Maf-expressing embryos (,,,). However, it is apparent that L-Maf did induce δ-crystallin maximally (). In most cases, MafB overexpression hampered lens shape (,). A section of the embryo shown in revealed a lack of lens invagination and proper arrangement of cells (). Nonelectroporated contralateral eye section showed normal expression of δ-crystallin and proper lens development (). In similar experiments, cryosections from the eclectroporated embryos were subjected to in situ hybridization using Dig-labeled CP49 probe (-). L-Maf (,) strongly induced CP49, while c-Maf (,) and MafB (,) showed lower activation of CP49. Further, whole-mount in situ hybridization was performed using L-Maf electroporated embryos after 5 h of incubation (,; stage 11), which revealed ectopic CP49 expression (; arrow). Green fluorescence of GFP indicates transgene expression (,,,,,,,,). The figure is representative of at least three independent experiments. For whole-mounts, five to six embryos were used each time.<p><b>Copyright information:</b></p><p>Taken from "Sequential and combinatorial roles of family genes define proper lens development"</p><p></p><p>Molecular Vision 2007;13():18-30.</p><p>Published online 16 Jan 2007</p><p>PMCID:PMC2503189.</p><p></p

Whole-mount and cryosection in situ hybridization exhibit ectopic expression of Six3 when L-Maf, but not c-Maf and MafB, is misexpressed

Electroporated chick embryos with L-Maf (-), c-Maf (-), MafB (-), and empty vector (,) were analyzed whole and in sections at stage 16 to follow the expression of Six3. Ectopic Six3 was detectable in the surface ectoderm expressing exogenous L-Maf (; yellow box shown in the inset; ; yellow arrows), but undetectable in c-Maf (,) and MafB (,) expressing cells outside lens. Contralateral nonelectroporated eyes showed normal expression of Six3 (,,). Embryo electroporated with empty vector as control showed only endogenous expression of Six3 (). Green fluorescence of GFP depicts electroporated cells (1st and 3rd columns). This figure is representative of at least three independent experiments. For whole-mounts, five to six embryos were used each time.<p><b>Copyright information:</b></p><p>Taken from "Sequential and combinatorial roles of family genes define proper lens development"</p><p></p><p>Molecular Vision 2007;13():18-30.</p><p>Published online 16 Jan 2007</p><p>PMCID:PMC2503189.</p><p></p

A diagram showing gene regulation within the family and of other effector genes during lens development in the chick

The bold arrow indicates the order of expression for the three large genes in lens lineage. An early-expressed Maf positively regulates later-expressed members. Later-expressed MafB inhibits the expression of L-Maf and c-Maf. All three Maf proteins can activate several common genes such as , , , [], (H.M.R. and K.Y., unpublished data), and . On the other hand, distinct effects are also observed, as L-Maf induces Six3, Cx43, and Cx45.6; c-Maf upregulates Cx43, and MafB induces E-cadherin but downregulates Cx43. Hence, both redundant and distinct functions of L-Maf, c-Maf, and MafB essentially control many vital genes during lens development.<p><b>Copyright information:</b></p><p>Taken from "Sequential and combinatorial roles of family genes define proper lens development"</p><p></p><p>Molecular Vision 2007;13():18-30.</p><p>Published online 16 Jan 2007</p><p>PMCID:PMC2503189.</p><p></p

Additional file 3 of Association between the efficacy and immune-related adverse events of pembrolizumab and chemotherapy in non-small cell lung cancer patients: a retrospective study

Additional file 3: Supplementary figure S2. Severe irAEs and efficacy. Kaplan–Meier curves for progression-free survival in patients with irAEs of grade 3 or higher versus patients without irAEs

Additional file 2 of Association between the efficacy and immune-related adverse events of pembrolizumab and chemotherapy in non-small cell lung cancer patients: a retrospective study

Additional file 2: Supplementary figure S1. IrAEs and survival. Kaplan–Meier curves for overall survival in patients treated with pembrolizumab and chemotherapy (a) and those treated with pembrolizumab monotherapy (b)

Additional file 1 of Association between the efficacy and immune-related adverse events of pembrolizumab and chemotherapy in non-small cell lung cancer patients: a retrospective study

Additional file 1: Supplementary Table S1. Univariable and multivariable time-varying Cox model analysis of covariates for progression-free survival in the combination therapy and monotherapy groups

IGF1R was phosphorylated on hypoxic GRPs, and knockdown of IGF1 decreased the population of CD133- and Oct4-positive hypoxic GRPs.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for IGF1 in PC9 or HCC827 parental cells, GRPs, and hypoxic GRPs. Data were normalized to actin expression. **p<0.001. <b>B.</b> PC9 cells, grown on Lab-Tek chamber slides with or without 1 µM gefitinib and under normoxic or hypoxic conditions for 72 h, fixed, and incubated with the primary antibodies against phosphorylated IGF1R (pIGF1R) and then with secondary antibodies labeled with Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained using an Axioplan 2 system imaging with AxioVision software. Images used to compare PC9 parental cells, GRPs, and hypoxic GRPs were acquired with the same instrument settings and exposure times, and were processed similarly. The number of pIGF1R-positive cells was counted, and the ratio of these cells was calculated in five fields in each experiment. **p<0.001. <b>C.</b> IGF1 expression was knocked down in PC9 or HCC827 hypoxic GRPs by using small interfering RNA (siRNA) in Lab-Tek chamber slides. Immunofluorescence staining for pIGF1R, CD133, or Oct4 was then performed. Two specific siRNAs and one non-specific control were used. The numbers of pIGF1R-, CD133- and Oct4-positive cells were counted, and the ratio of these cells was calculated in five fields for each experiment. **p<0.001, *p<0.01.</p

Tumor incidence of parental PC9 cells and normoxic and hypoxic GRPs transplanted into NOG mice.

<p>GRPs; Gefitinib resistant persisters.</p><p>NOG mice; NOD/Shi-scid/IL-2Rcnull (NOG) mice.</p>#<p>Tumor incidence was increased significantly in hypoxic GRPs group as compared with parental cell group with 1×10¹ cells/injection, p<0.05.</p><p>To evaluate the <i>in vivo</i> tumorigenic potential, 1×10 cells or 1×10² cells of parental PC9 cells or normoxic and hypoxic PC9 GRPs were mixed with Matrigel and injected into both flanks of NOG mice. Tumor formation was evaluated 33 days after injection.</p

Sphere-forming ability of GRPs was upregulated and hypoxia increased this capacity.

<p>PC9 parental cells, GRPs, and hypoxic GRPs were prepared at densities of 2.5×10³ cells per 2 mL per well in serum-free media supplemented with growth factors and seeded into 6-well ultra-low attachment plates. PC9 parental cells and GRPs were incubated under normoxic conditions, while PC9 hypoxic GRPs were grown under hypoxic conditions. Culture medium was fed every 3 days. The number and size of spheres were recorded and immunofluorescence was performed 7 days after the start of the culture period. Spheres were fixed and incubated with primary antibodies against CD133, Oct4, or phosphorylated IGF1R (pIGF1R), and then with secondary antibody labeled with Alexa Fluor 594 goat anti-mouse IgG (red) or Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained on an Axioplan 2 imaging system with AxioVision software. <b>A.</b> The number of spheres was significantly increased in PC9 GRPs compared to in parental cells, and was further increased in PC9 hypoxic GRPs. *p<0.01, # p<0.05. <b>B.</b> Sphere size of PC9 hypoxic GRPs was significantly greater than that of parental cells. # p<0.05. <b>C.</b> Immunofluorescent images of control cells of PC9 (left) and spheres of GRPs (right) for CD133, Oct4, and pIGF1R.</p

GRPs were highly enriched for gene expression of stemness, IGF1, and IGF1R.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1, which are stemness genes in PC9 or HCC827 parental cells and GRPs. <b>B.</b> Quantitative RT-PCR was performed with primers specific for IGF1 and IGF1R in PC9 or HCC827 parental cells and GRPs. Data were normalized to actin expression. *p<0.01, **p<0.001, ***p<0.0001.</p