EMBO/soda-nel huggingface dataset:
Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice (2022)
Figure 7

Figure 7. CLAP regulates itch-related gene expression by partially through the regulatory role of MSI2

(A) Research flowchart for RNA-seq of DRGs following intrathecal injection.

(B) Visualization of knockdown efficiency of CLAP in RNA-seq from UCSC genome browser with customized tracks. RNA-seq were collected from siNC (n = 3) and siCLAP (n = 3) injected mice.

(C) The expression changes of genes upon CLAP knockdown by RNA-seq. Differently expressed genes were selected by foldchange ≥ 1.2 and P ≤ 0.05. Red, blue, green and gray dots represent upregulated, downregulated, downregulated C2-specific and unchanged genes, respectively.

(D) The expression changes of C2-specific and itch-related genes upon CLAP knockdown by RNA-seq. Each dot represents an RNA-seq sample, and results are presented as mean ± SEM (n = 3). * P < 0.05, ** P < 0.01 and *** P < 0.001 versus siNC.

(E) Research flowchart for AAV-mediated overexpression of CLAPR by intrathecal injection of AAV-CLAPR.

(F) qPCR showed that downregulated CLAP and C2-specific genes including Sst, Plcb3 and Cysltr2 by siCLAP was fully rescued by overexpression of CLAPR in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 10; siCLAP, n = 6; siCLAP + CLAPR, n = 12). * P < 0.01 versus siNC and # P < 0.05, ## P < 0.01 versus the indicated treatment.

(G) qPCR showed the association of CLAP with MSI2, but not for Sst in mouse DRG following MSI2 RIP experiment. The results are presented as mean ± SEM (n = 3). * P < 0.05 verus IgG.

(H) Research flowchart for AAV-mediated overexpression of CLAPR by intrathecal injection of AAV-CLAPR.

(I) qPCR showed that downregulated Plcb3 by siCLAP was fully rescued by knockdown of MSI2 in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 8; siCLAP, n = 6; siCLAP + siMSI2, n = 5). * P < 0.05, *** P < 0.001 versus siNC and ## P < 0.01 versus the indicated treatment.

Data information: All statistical analysis were used Unpaired T-Test.

Expanded View Figure Legends
Selective depletion of metastatic stem cells as therapy for human colorectal cancer (2018)
Figure 1

Figure 1. T22-GFP-H6-FdU nanoconjugate synthesis and selective internalization and killing of CXCR4+ CRC cells in vitro

(A) The nanoconjugate contains a fusion protein [T22-GFP-H6 -composed of the peptide T22 as a CXCR4 ligand, a green fluorescent protein and a Histidine tail - bound to the payload drug (Unzueta et al,, 2012)].

(B) Three to four pentameric oligonucleotides (approximately 20 molecules) of the antitumor drug 5-Fluoro-2'-deoxyUridine (FdU), named oligo-FdU, are conjugated to the T22-GFP-H6 targeting vector using a linker.

(C) T22-GFP-H6-FdU chemical synthesis: T22-GFP-H6 is first covalently bound to the 6-Maleimidohexanoic acid N-hydroxysuccinimide ester linker through its amino groups in the external lysines (Hermanson et al, 2013). The thiol-functionalized oligo-FdU (oligo-(FdU)5-SH) (see Appendix Fig S1) is then reacted with T22-GFP-H6 functionalized with maleimide (Michael reaction).

(D) High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry.

(E) Lack of human SDF-1α release from cultured SW1417 CRC cells, as measured by ELISA, whereas human control 1BR3.G fibroblasts express high SDF-1α levels, after 48h or 72h of growth in culture. (N=2 experiment in duplicate)

(F) Nanoconjugate internalization in CXCR4 overexpressing (CXCR4+) SW1417 CRC cells after 1 hour exposure at 1µM, as measured by fluorescence emission using flow cytometry. (N=3 experiments in duplicate) Significant difference at p= 0.002 between T22-GFP-H6-FdU and AMD3100+T22-GFP-H6-FdU groups

(G) Intracellular trafficking of T22-GFP-H6-FdU in CXCR4+ SW1417 cells by confocal microscopy after exposure at 1µM for 24 h. The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye (CellMask™), whereas cell nucleus was stained in blue with hoescht. The insets show detail of the intracellular localization of nanostructured, fluorescent entities, in an isosurface representation within a three-dimensional volumetric x-y-z data field

(H) Linearized T22-GFP-H6-FdU dose-response trend line representation compared with unconjugated free oligo-FdU exposure. Antitumor effect was measured as CXCR4+ SW1417 cell viability by MTT after 72 h exposure as the described concentrations. (N=3 experiments in duplicate)

(I) Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22-GFP-H6-FdU for 48 h, as compared to T22-GFP-H6 or free oligo-FdU. (N=3 experiments in duplicate; Scale bar, 100µm).
Selective depletion of metastatic stem cells as therapy for human colorectal cancer (2018)
Figure 2

Figure 2. Selective biodistribution and receptor-dependent uptake of T22-GFP-H6-FdU in CXCR4+ cells in vivo

(A) Approach to achieve targeted drug delivery and selective killing of metastatic stem cells: CXCR4-nanoconjugate interaction triggers CXCR4-mediated internalization in MetSCs, in primary tumors and metastatic foci, followed by FdU release to the cytosol and diffusion to the nucleus to induce double stand breaks leading to selective killing of CXCR4+ cells.

(B) Selective T22-GFP-H6-FdU nanoconjugate biodistribution in subcutaneous CXCR4+ SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose, as measured by fluorescence emission using IVIS Spectrum 200 (N=5/group). Biodistribution is similar to that achieved by the T22-GFP-H6 targeting vector and undetectable after Buffer or free oligo-FdU treatment. (N=5 mice/group)

(C) Co-localization (yellow merged) of the T22-GFP-H6-FdU (green) and the CXCR4 receptor (red) and release of T22-GFP-H6-FdU into the cytosol in CXCR4+ tumor cells 5h after a 100ug dose of nanoconjugate, as measured by dual anti-GFP/anti-CXCR4 immunofluorescence (IF). DAPI (blue nuclear staining). Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area (A)±s.e.m (µm2) (N=10, 2 tumor fields x 5 mice; 200X). Note the significant (P=0.003) increase in the area occupied by the green dots (nanoconjugate realeased to the cell cytosol) in T22-GFP-H6-FdU-treated tumors, compared to free oligo-FdU-treated control tissues. Scale bar, 50 µm.

(D) Administration of the CXCR4 antagonist AMD3100 completely blocks T22-GFP-H6-FdU tumor biodistribution, as measured by fluorescence emission. Fluorescence is not detected in Buffer or free oligo-FdU controls (N=5 tumor fields/group).

(E) The uptake of T22-GFP-H6-FdU observed in CXCR4+ SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration, as quantified using the anti-GFP IHC H-score. (N=5 tumor fields/group).

Comparison of T22-GFP-H6 uptake between groups: (B:Buffer; F:Free oligo-FdU; T-F: T22-GFP-H6-FdU; T-F+A:T22-GFP-H6-FdU+AMD3100):

P values for Statistical Differences **B vs T-F, P=0.000; **F vs T-F, P=0.000; **T-F vs TFA, P=0.004.

(F) Representative images of T22-GFP-H6-FdU uptake and AMD3100 competition by anti-GFP inmunostaining, which quantitation is reported in panel E. Scale bar, 50 µm
Selective depletion of metastatic stem cells as therapy for human colorectal cancer (2018)
Figure 3

Figure 3. T22-GFP-H6-FdU-induced depletion of CXCR4-overexpressing cancer cells in tumor tissue

(A) Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N=5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6 and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase 3 activation (measured with anti-ɣH2AX or anti-cleaved caspase 3 by IHC) 5h post-administration (middle panels). Apoptotic induction (Hoescht staining, 24h post-administration, lower panels). Note the higher number of cells positive for DSBs, Caspase 3 activation and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells.

(B) Quantitation of the number of cells containing DSBs or active Caspase-3 in IHC-stained tumor sections 5h post-treatment and the number of condensated or disaggregated nuclei (by Hoescht staining) 24h post-treatment in tumor sections of 10 high-powder fields (400x magnification) using the Cell D software. (N=50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean±s.e.m.

Parameter comparison between groups: (B: Buffer; T: T22-GFP-H6; F: Free oligo-FdU; T-F: T22-GFP-H6-FdU). P values for Statistical Differences:

-ɣ-H2AX staining quantitation: #B vs T, P=0.001; #B vs F, P=0.000; #B vs T-F, P=0.000; **T vs T-F, P=0.001; *F vs T-F, P=0.02

-Cleaved caspase 3 quantitation: *B vs F, P=0.034; **B vs T-F, P=0.009; **T vs T-F, P=0.003; *F vs T-F, P=0.012

-Hoescht staining quantitation: **B vs F, P=0.01; **B vs T-F, P=0.001; **T vs T-F, P=0.000; *F vs T-F, P=0.032
Selective depletion of metastatic stem cells as therapy for human colorectal cancer (2018)
Figure 4

Figure 4. T22-GFP-H6-FdU-induced depletion of CXCR4-overexpressing cancer cells in tumor tissue leading to reduced spheroid formation capacity

(A, B) T22-GFP-H6-FdU depletes CXCR4+ cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration. Note the reduction in CXCR4+ cell fraction in the tumor 24h after injection, their almost complete elimination at 48h and the re-emergence of CXCR4+ cells 72h post-administration, using anti-CXCR4 IHC. In contrast, the CXCR4+ cancer cell fraction (CXCR4+ CCF) in tumor tissue remains constant along time after free oligo-FdU treatment. The three day time-lapse for CXCR4+ tumor cell re-appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect. (N=5: 5 mice/group; 1 samples/mouse). Scale bar, 50 µm. Data expressed as mean±s.e.m

CXCR4 H-score comparison for T22-GFP-H6-FdU(T-F)-treated tumors among time points (Green line, panel A); P values for Statistical Differences: *T-F Basal vs T-F 24h, P= 0.038; **T-F Basal vs T-F 48h, P=0.001; **T-F Basal vs T-F 72h, P=0.003; *T-F 24h vs T-F 48h, P=0.033

CXCR4 H-score comparison between T22-GFP-H6-FdU (T-F) and Free oligo-FdU (F) (Black line, panel A); P values for Statistical Differences: **T-F vs F at 48h, P=0.001; T-F vs F at 72h, *P=0.034).

(C-D) Significant reduction in the number of spheroid formed (C. optical microscope) and their bioluminescence emission (D, IVIS Spectrum 200), generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates), obtained from CXCR4+ luciferase+ SW1417 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. D. Quantitation of the bioluminescent signal (BLI) expressed as Average Radiant Intensity, obtained using the IVIS spectrum 200 equipment (N=2 plates/group). Data expressed as mean±s.e.m. Comparison of emitted BLI between groups: (B: Buffer; F: Free oligo-FdU; T-F: T22-GFP-H6-FdU). P values for Statistical Differences: **T-F vs B, P=0.001 (Green line, panel D); *F vs B, P=0.011 (Red line); *T-F vs F at P= 0.02 (Black line, panel D).
Selective depletion of metastatic stem cells as therapy for human colorectal cancer (2018)
Figure 5

Figure 5. T22-GFP-H6-FdU-induced reduction of tumor re-initiation capacity and blockade of tumor emboli intravasation in the CXCR4+ patient-derived M5 model

(A-B). Reduction in the number of formed spheroids (optical microscope) generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates) obtained from CXCR4+ M5 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. (N=8; 2 mice/group; 4 plates/mouse). (Scale bar, 100µm). Comparison of spheroid formation between groups: (B: Buffer; F: Free oligo-FdU; T-F: T22-GFP-H6-FdU). P values for Statistical Differences: **T-F vs B, P=0.001 (Green line, Panel B); *F vs B, P=0.012 (Red line); **T-F vs F, P= 0.001 (Black line, Panel B).

(C-D). Reduction in tumor re-initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice (N=4 tumors/group) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days , as compared to Free oligo-FdU-treated or Buffer-treated mice. Recording of the number and size of positive tumors. (N=4; 2 mice/group, 2 injection points/mouse).

(E-G) Representative images of tumor emboli intravasation determined by microscopic analyses of H&E-stained tumor sections (N=5/group).

(F) T22-GFP-H6-FdU-induced reduction in the number of intravasated tumor emboli in peri-tumoral vessels of the M5-orthotopic primary tumor (E. optical images; F. emboli number quantitation) and reduction of CXCR4 expression in these emboli (G), treated 7 days after tumor cell implantation with 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. Tumor emboli counting in 10 high powder field at 200x magnification in H&E stained sections from each tumor. (N=5/group). Data expressed as mean±s.e.m.

Comparison of tumor emboli number between groups: (B: Buffer; F:Free oligo-FdU; T-F: T22-GFP-H6-FdU). P values for Statistical Differences: **T-F vs B, P=0.038 (Green line, Panel F), *T-F vs F, P=0.016 (Black line, Panel F) (G) CXCR4 expression per tumor emboli determined by using anti-CXCR4 IHC and calculating H-score (multiplying percent of CXCR4+ cells out of total cell number in the emboli area by their staining intensity, scoring each from 0 to 3 (where 3 is the maximal intensity) per tumor emboli area. (N=5 mice/group). Comparison of CXCR4 H-score between groups: (B: Buffer; T-F: T22-GFP-H6-FdU). P values for Statistical Differences: T-F vs B at P=0.027 (Green line, Panel G)
Selective depletion of metastatic stem cells as therapy for human colorectal cancer (2018)
Figure 7

Figure 7. Negligible T22-GFP-H6-FdU biodistribution or toxicity on non-tumor tissues

(A) Undetectable T22-GFP-H6-FdU emitted fluorescence in normal tissues, except for a transient accumulation 5h after a 100μg dose in the liver, which disappears at 24 h. Liver emitted fluorescence is transient and significantly lower than the one registered in tumor tissue. Tumor/Liver ratio= 7.5 (see tumor intensity in Fig 2B, which was registered in the same experiment) (N=5 mice/group). Scale bar, 1 cm.

(B) Representative images depicting the level of DNA double strand break (DSB) induction in histologically normal bone marrow 5h after treatment, as measured by anti-ɣ-H2AX, which is higher in free oligo-FdU-treated mice than in T22-GFP-H6-FdU (P=0.047). Low level of cells containing DSBs in histologically normal kidney after T22-GFP-H6-FdU or free oligo-FdU treatment, a finding occurring in all normal tissues analysed. (N=50, 5 mice/group; 10 fields/mouse).

(C) Representative images showing lack of histopathological alterations in H&E stained tissue or apoptotic induction in H&E stained samples of CXCR4+ (bone marrow) and CXCR4- (brain, kidney, liver, lung and heart) normal tissues 24h after the administration of a 100 μg dose of T22-GFP-H6-FdU or an equimolecular dose of free oligo-FdU (N=5/group). Note that the transient nanoconjugate distribution to liver or the DNA damage induced in bone marrow do not lead to cytoxicity on these no-tumor tissues. (N=50, 5 mice/group; 10 fields/mouse)

(D) Lack of differences in body weight among groups registered along time in the SW1417-derived CCR model and the regression of metastases protocol (N=10 mice/group).

(E) Lack of differences in body mouse weight among groups registered along time in the SW1417 cell line-derived model and the prevention of metastasis protocol (Buffer (N=11; Free oligo-FdU (N=12), T22-GFP-H6-FdU (N=12)).

(F) Lack of differences in body mouse weight among groups registered along time in the M5 patient-derived model and the prevention of metastasis protocol (Buffer (N=6); Free oligo-FdU (N=17); T22-GFP-H6-FdU (N=8). Scale bar, 100 μm
NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system (2019)
Figure 1

Figure 1. NBR1 promotes an increase in p62-levels, phosphorylation and body formation. (A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP or NBR1 under the CAG promoter for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.

(C) Immunofluorescence microscopy. Wild type hepatocytes were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.

(D) FRAP assay. Wild type hepatocytes cultured in glass-bottom plates were infected with GFP-NBR1 adenovirus for 48 hrs. The signal recovery after photobleaching was measured, quantified from at least three independent biological replicates, and is represented as means ± s.e. Bar: 1 µm.
NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system (2019)
Figure 2

Figure 2. NBR1 increases protein and mRNA levels of Nrf2 targets through p62.

(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP-Nrf2 and GFP or NBR1 for 48 hrs. Total cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. **P < 0.01 as determined by Welch's t-test.

(B) Immunoblot analysis. Primary hepatocytes prepared from p62f/f and p62f/f; Alb-Cre mice were infected with adenovirus against GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.

(C) Immunofluorescence microscopy. Primary hepatocytes from wild type mice were infected with adenovirus expressing GFP-Nrf2 and LacZ or NBR1 for 48 hrs. More than 100 cells were assayed for the nuclear signal of Nrf2. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test. Bars: 10 µm.

(D) Total RNAs were prepared from primary hepatocytes described in (B). Values were normalized against the amount of mRNA in the p62f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.
NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system (2019)
Figure 3

Figure 3. Phase-separation and phosphorylation of p62 are needed for full activation of Nrf2 mediated by NBR1.

(A) Immunofluorescence microscopy. Primary hepatocytes prepared from p62f/f mice were infected with or without adenovirus Cre recombinase for 48 hrs and then additionally infected with adenovirus LacZ or NBR1. 24 hrs after the infection, the hepatocytes were immunostained with anti-Keap1, anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 10 µm.

(B) Immunoprecipitation analysis. Crude extracts were prepared from primary hepatocytes as described in (A) and then immunoprecipitated with anti-FLAG (as a negative control) or anti-NBR1 antibody. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three separate experiments.

(C) Primary hepatocytes prepared from p62f/f; Alb-Cre mice were co-infected with adenovirus GFP or NBR1 in combination with wild-type p62 or the mutants for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.

(D) RT qPCR analysis. Total RNAs were prepared from hepatocytes described in (C). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.
NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system (2019)
Figure 4

Figure 4. NBR1 enhances the protein levels of p62 partially independent on Nrf2.

(A) Immunoblot analysis. Primary hepatocytes prepared from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nrf2f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.

(C) Immunofluorescence microscopy. Hepatocytes isolated from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.
NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system (2019)
Figure 5

Figure 5. NBR1 prevents the autophagic degradation of p62.

(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 (BafA1) for 24 hrs or cultured in amino acid-deprived medium (Stv) for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.

(B) Fluorescence microscopy. p62-deficient MEFs were co-transfected with mCherry-GFP-p62 in combination with wild-type NBR1 or NBR1 D50R mutant. 48 hrs after the transfection, fluorescence images were observed. Bars: 10 µm. The number of puncta positive for only red-signal in each MEFs was counted in more than 100 cells. Bar graphs are represented as means ± s.e. ***P < 0.001 as determined by Welch's t-test.