Figure 1.
Effects of d.n. IKKβ-EGFP on muscle fiber cross sectional area (CSA).
(A) Mean tibialis anterior (TA) fiber cross-sectional area in control and C26 tumor-bearing mice in the presence or absence of d.n. IKKβ-EGFP. Each bar represents mean fiber area from 8 muscles (± SE). The average number of fibers measured per muscle was 200. (B) Fiber area frequency distribution of all fibers from muscles of control, C26, and C26 + d.n. IKKβ-EGFP groups. (C) Western blots of lysates from TA muscles injected with the empty vector (EV) or d.n. IKKβ-EGFP plasmid confirms overexpression of the fusion protein. d.n. IKKβ-EGFP is 114 kDa. All samples are from the same immunoblot. (D) Representative cross sections of TA muscles from control and C26 tumor-bearing mice injected with the d.n. IKKβ-EGFP plasmid. Fluorescent red represents laminin staining (anti-laminin incubation followed by Texas Red-X fluorescent dye-conjugated secondary antibody). Fluorescent green cytoplasm represents expression of transfected d.n. IKKβ-EGFP. C = control; * significantly different from control (P<0.05). † significantly different from C26 fibers not transfected with d.n. IKKβ-EGFP (P<0.05).
Figure 2.
Effects of IκBαSR-EGFP on muscle fiber cross sectional area (CSA).
(A) Mean tibialis anterior (TA) fiber cross-sectional area in control and C26 tumor-bearing mice in the presence or absence of IκBαSR-EGFP. Each bar represents mean fiber area from 8 muscles (± SE). (B) Fiber area frequency distribution of all fibers from muscles of control, C26, and C26 + IκBαSR-EGFP groups. (C) Western blots of lysates from TA muscles injected with the empty vector (EV) or IκBαSR-EGFP plasmid confirms overexpression of the fusion protein. IκBαSR-EGFP is 64 kDa. All samples are from the same immunoblot. (D) Representative cross sections of TA muscles from control and C26 tumor-bearing mice injected with the IκBαSR-EGFP plasmid. C = control. * significantly different from control (P<0.05). † significantly different from C26 fibers not transfected with IκBαSR-EGFP (P<0.05).
Figure 3.
Effects of d.n.IKKα-EGFP on muscle fiber cross sectional area (CSA).
(A) Mean tibialis anterior (TA) fiber cross-sectional area in control and C26 tumor-bearing mice in the presence or absence of d.n. IKKα-EGFP. Each bar represents mean fiber area from 8 muscles (± SE). (B) Fiber area frequency distribution of all fibers from muscles of control, C26, and C26 + d.n. IKKα-EGFP groups. (C) Western blots of lysates from TA muscles injected with the empty vector (EV) or d.n. IKKα-EGFP plasmid confirms overexpression of the fusion protein. d.n. IKKα-EGFP is 108 kDa. All samples are from the same immunoblot. (D) Representative cross sections of TA muscles from control and C26 tumor-bearing mice injected with the d.n. IKKα-EGFP plasmid. C = control. * significantly different from control (P<0.05).
Figure 4.
Effects of d.n. NIK on muscle fiber cross sectional area (CSA).
(A) Mean tibialis anterior (TA) fiber cross-sectional area in control and C26 tumor-bearing mice in the presence or absence of d.n. NIK. Each bar represents mean fiber area from 8 muscles (± SE). (B) Fiber area frequency distribution of all fibers from muscles of control, C26, and C26 + d.n. NIK groups. (C) Western blots of lysates from TA muscles injected with the empty vector (EV) or d.n. NIK plasmid confirms overexpression of the protein. d.n. NIK = 130 kDa. All samples are from the same immunoblot. (D) Representative cross sections of TA muscles from control and C26 tumor-bearing mice injected with the d.n. NIK plasmid (encodes GFP on separate cistron). C = control. * significantly different from control (P<0.05).
Figure 5.
Effects of d.n.p65-EGFP on muscle fiber cross sectional area (CSA).
(A) Mean tibialis anterior (TA) fiber cross-sectional area in control and C26 tumor-bearing mice in the presence or absence of d.n. p65-EGFP. Each bar represents mean fiber area from 8 muscles (± SE). (B) Fiber area frequency distribution of all fibers from muscles of control, C26, and C26 + d.n. p65-EGFP groups. (C) Western blots of lysates from TA muscles injected with the empty vector (EV) or d.n. p65-EGFP plasmid confirms overexpression of the fusion protein. (D) The d.n. p65(313)-EGFP fusion protein (313 a.a.+238 a.a. = 551 a.a. = 63 kDa) is of similar molecular weight as endogenous mouse whole p65 protein (549 a.a. = 60.2 kDa) and they are not separable on a 4-15% gradient polyacrylamide gel. All samples are from the same immunoblot. (E) Representative cross sections of TA muscles from control and C26 tumor-bearing mice injected with the d.n. p65-EGFP plasmid. C = control. * significantly different from control (P<0.05). † significantly different from C26 fibers not transfected with d.n.p65-EGFP (P<0.05).
Figure 6.
Effects of d.n. c-Rel-EGFP on muscle fiber cross sectional area (CSA).
(A) Mean tibialis anterior (TA) fiber cross-sectional area in control and C26 tumor-bearing mice in the presence or absence of d.n. c-Rel-EGFP. Each bar represents mean fiber area from 8 muscles (± SE). (B) Fiber area frequency distribution of all fibers from muscles of control, C26, and C26 + d.n. c-Rel-EGFP groups. (C) Western blots of lysates from TA muscles injected with the empty vector (EV) or d.n. c-Rel-EGFP plasmid confirms overexpression of the fusion protein. d.n.c-Rel-EGFP is 60 kDa. All samples are from the same immunoblot. (D) Representative cross sections of TA muscles from control and C26 tumor-bearing mice injected with the d.n.p65-EGFP plasmid. C = control. * significantly different from control (P<0.05).
Figure 7.
Gel shift assay of NF-κB binding and NF-κB reporter activity.
(A) Bands represent protein-oligonucleotide binding of gastrocnemius nuclear extracts from control and C26 mice 23 days post-inoculation. 20 µg of protein incubated with infrared dye labeled NF-κB oligonucleotide. A lane labeled “Cold” indicates incubation with 200X unlabeled consensus oligonucleotide competitor. Competitor lane was on same gel as lanes 1 through 4. (B) Signal intensity of bands representing protein binding to NF-κB oligonucleotide; control mean value represents 12 independent samples, C26 mean value represents 14 independent samples. Values were combined from 13, 17 and 23 days post-tumor cell inoculation since there was no difference in binding compared to controls at any time point. (C) As a positive control for the NF-κB gel shift assay, nuclear extracts isolated from hind limb muscles of 42 day old Lama2−/− mice compared to aged matched C57BL/6 wild type (WT) controls are shown. 15 µg of protein incubated with infrared dye labeled NF-κB oligonucleotide. + indicates incubation with 150X unlabeled consensus oligonucleotide competitor. Each lane represents an independent muscle sample. (D) NF-κB-dependent reporter activity in TA muscles from control and C26 tumor-bearing mice. NF-κB-dependent luciferase activity in TA muscles at 12 and 25 days after tumor cell inoculation representing mild and severe cachexia. The number of muscles per group was 6 for the 12-day time point and 8 for the 25-day time point.
Figure 8.
Heatmaps of selected upregulated genes in plantar flexor muscles from several functional categories.
Data are from muscles at 25 days post-tumor inoculation (severe cachexia). Red color in the heatmap represents gene upregulation in muscles from C26 mice compared to the lower control values represented by blue. All genes represented are increased greater than 1.5-fold. Genes involved in: ubiquitin-proteasomal protein degradation, autophagy, other proteolytic processes (e.g. caspases, cathepsins, and metalloproteases), immune responses (innate and adaptive), and transcription factor genes are shown. The heatmaps capture most of the upregulated genes in these functional categories but do not represent a complete list (see Table S2). Each color block represents one pooled sample (control n = 3; C26 n = 3).
Table 1.
Enriched GO terms in muscle of C26 mice using DAVID functional annotation.
Figure 9.
ChIP PCR to validate the p65 antibody and comparison of p65 binding in cancer cachexia vs. a classic inflammatory system.
(A) Chromatin was prepared from C2C12 myotubes that had been untreated or treated with 10 ng/ml mouse TNF for 4 hours and from (B) gastrocnemius/plantaris muscles of mice with and without 25 days of C26 tumor-induced cachexia. Chromatin was sonicated and immunoprecipitated without antibody or with the same antibody to p65 used for ChIP-seq or with a p50 antibody. Captured precipitates were used to prepare DNA template evaluated by PCR with primers that flank two closely spaced validated NF-κB sites in the IP-10 promoter. PCR products are shown on acrylamide gels, stained with Sybr gold. Standards are 100 bp ladder. Input lanes are PCR products using the same primers on chromatin taken before ChIP.