Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN in brain sections from 16-month-old mice WT, TDP-43 Q331K/Q331K (Q331K), Grn −/− , TDP-43 Q331K/Q331K Grn −/− (Q331K Grn −/− ) mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43, phosphorylated TDP-43 (pS409/410), and PGRN levels in cortical lysates from 16-month-old mice of the indicated genotypes. C TDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3). E Total RNAs were extracted from the cortex of 10-month-old WT, TDP-43 Q331K/Q331K , Grn −/− , TDP-43 Q331K/Q331K Grn −/− male mice, and the RT-qPCR was performed to analyze the splicing changes in Sort1 exon 17b (left) and Mapt exons 2 and 3 (right). The relative mRNA levels of transcripts including or excluding exons 2 and 3 represent the inclusion of Mapt exons 2 and 3. Data are presented as mean ± SEM ( n = 4 mice per genotype). p -values were determined using one-way ANOVA tests with Bonferroni’s multiple comparisons. F Expression levels of Tardbp in WT and Q331K mice. Total RNAs were extracted from the cortex of 10-month-old WT and Q331K male mice, and the RNA-seq was performed to analyze gene expression changes. Normalized read counts are shown. Data are presented as mean ± SEM ( n = 5-6 mice per genotype). * p < 0.05, unpaired two-tailed Student's t-test.

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining, Quantitative RT-PCR, Expressing, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN using rabbit anti-TDP-43 CTD antibodies and mouse anti-NeuN antibodies, respectively (left panel) or immunostaining of human TDP-43 (right panel) using mouse anti-human TDP-43 in brain sections from 21-day-old WT, hTDP-43 Tg/Tg (Tg/Tg), Grn −/− , and hTDP-43 Tg/Tg Grn −/− mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43 and phosphorylated TDP-43 (pS409/410) and PGRN levels in brain lysates from 21-day-old mice. Antibodies recognizing the C-terminal or N-terminal domain of TDP-43 were used to detect total TDP-43 levels. C TDP-43 and pTDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. ** p < 0.01. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3).

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining

Journal: Microbiology Spectrum

Article Title: High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing Effects

doi: 10.1128/spectrum.03760-22

Figure Lengend Snippet: Construction of cytosine base editor (CBE)-STOP for genetically engineering C. glutamicum and the features of pCoryne-BE3. (a) Scheme of CBE using pCoryne-BE3 and pCoryne2-single-guide RNA (sgRNA) in C. glutamicum . After DNA replication or repair, a C•G-to-T•A conversion occurred. CBE-STOP generates stop codons (TGA, TAG, or TAA) in the coding strand sequence by converting the targeted C to T of C AA (Gln), C AG (Gln), or C GA (Arg) in the coding strand and in the noncoding strand of T GG (Trp) at the editing window. The protospacer adjacent motif (PAM) site for the CBE-STOP was 5′-NGG. (b) Targetable coding sequences by CBE-STOP with at least one stop codon in the C4 to C8 position (Fig. S1) using either 5′-NGG PAM or 5′-NG PAM. (c) Mutation frequencies (%) or base editing performance (%) at the editing-window positions. Points in bars represents each mutation frequency for eight different protospacers at editable window (C3 to C12). See the detail data (Table S6). (d) Mutation frequencies (%) for sequence-context dependency. C4 positions were only available with the GC motif. The error bar represents the standard deviation (SD). CDS, coding sequence; UGI, uracil glycosylase inhibitor; C. glutamicum, Coryebacterium glutamicum ; rAPOBEC1, rat cytidine deaminase; XTEN, a 16 amino acid flexible linker; nCas9, a DNA nickase; sgRNA, single guide RNA; Spc, spectinomycin; Cm, chloramphenicol.

Article Snippet: pgRNA-bacteria , ColE1 ( Ec ), Amp r , customizable guide RNA (gRNA), Addgene 44251 , .

Techniques: Sequencing, Mutagenesis, Standard Deviation

Journal: Microbiology Spectrum

Article Title: High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing Effects

doi: 10.1128/spectrum.03760-22

Figure Lengend Snippet: Bacterial strains and plasmids used ^a

Article Snippet: pgRNA-bacteria , ColE1 ( Ec ), Amp r , customizable guide RNA (gRNA), Addgene 44251 , .

Techniques: Plasmid Preparation, Cloning

Journal: Endocrinology

Article Title: Circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in metabolic syndrome.

doi: 10.1210/en.2014-1058

Figure Lengend Snippet: Figure 1. Serum PGRN concentrations and autophagic activity of omental AT in subjects with and without MS. A, Serum PGRN levels in cohort 1 subjects with (n 91) and without (n 67) MS. B, Serum PGRN levels stratified by the number of the components of MS (n 36, 33, and 22, respectively, in 3-, 4-, and 5-component groups). mRNA and protein expressions of PGRN or autophagy-related genes were measured in omental AT samples from the subjects of cohort 2 without (n 15) and with MS (n 17). C, Relative expression of PGRN mRNA examined by quantitative real-time PCR analysis in all groups normalized to the average levels of the mean-centered housekeeping gene GAPDH; the non-MS samples were set as 1. D, Representative Western blots of PGRN in subjects without and with MS. E, Serum PGRN levels in cohort 2 subjects. F, Relative expression of autophagy-related genes Atg7, LC3A, or LC3B normalized to GAPDH. G, Representative Western blots of Atg7, LC3-I/II, and p62 in AT. H, Univariate correlations between circulating PGRN and mRNA levels of autophagy genes in AT. I, ER stress markers XBP-1s and ATF6 were examined by quantitative real-time PCR analysis in omental AT samples obtained from the cohort 2 subjects. Results are presented as gene expression levels in all groups normalized to the average levels of the mean-centered housekeeping gene GAPDH. J, mRNA levels of IL-1 and IL-6. Data are means SEM in each bar graph. *, P .05 vs the non-MS group; **, P .001 vs the non-MS group. #, P .05 vs. the 3-component group.

Article Snippet: Cells were transfected with a silencing RNA (small interfering RNA [siRNA]; Santa Cruz Technology) targeted for human PGRN (sc-39261), NF- B-p65 (sc-29410), and XBP-1 (sc-38627) using Lipofectamine 2000 (Invitrogen). siRNA consisting of a scrambled sequence of similar length was used as a control.

Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Gene Expression

Journal: Endocrinology

Article Title: Circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in metabolic syndrome.

doi: 10.1210/en.2014-1058

Figure Lengend Snippet: Figure 4. Identification of the involvement of ER stress in PGRN-induced impaired insulin signaling and autophagy imbalance. A, Time-dependent effects of PGRN on ER stress markers and phosphorylation of PERK (p-PERK) and eIF2 (p-eIF2). B, Adipocytes were treated with 20 mM PBA in the presence of 100 ng/mL PGRN and then stimulated with insulin (10 nM) for 10 minutes. Phosphorylation of PERK, IRS-1 (pY), and Akt (p-Akt) and expression of Atg7 and LC3 were measured in adipocytes. C, Phosphorylation of PERK, IRS-1, and Akt and expression of Atg7 and LC3 with or without treatment with low-dose PGRN (20 ng/mL) for 16 hours in XBP-1/ adipocytes. The graph next to the blots shows the quantification of the protein levels under experimental conditions. A representative blot is shown, and all the data expressed as means SEM in each bar graph represent the average of at least 3 independent experiments. *, P .05 and **, P .01 vs the control (Ctrl); #, P .05 vs PGRN. IB, immunoblotting; IP, immunoprecipitation; siXBP-1, XBP-1 siRNA.

Article Snippet: Cells were transfected with a silencing RNA (small interfering RNA [siRNA]; Santa Cruz Technology) targeted for human PGRN (sc-39261), NF- B-p65 (sc-29410), and XBP-1 (sc-38627) using Lipofectamine 2000 (Invitrogen). siRNA consisting of a scrambled sequence of similar length was used as a control.

Techniques: Phospho-proteomics, Expressing, Control, Western Blot, Immunoprecipitation

Journal: Endocrinology

Article Title: Circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in metabolic syndrome.

doi: 10.1210/en.2014-1058

Figure Lengend Snippet: Figure 6. Effect of PGRN on ER stress, IR, and autophagy is mediated through the TNFR/NF-B pathway in adipocytes. For the effects of PGRN, adipocytes were treated with 100 ng/mL PGRN for 16 hours. For insulin signaling, cells were stimulated with 10 nM insulin for 10 minutes. Indicators of ER stress, insulin signaling, and autophagy were measured at protein levels. The relative quantity of proteins was analyzed with Quantity One software. A, Phosphorylation of PERK (p-PERK) and IRS-1 and expression of Atg7 in p55/, p75/, and p55/p75/ adipocytes. B, Human adipocytes were cultured in the presence or absence of 100 ng/mL PGRN with or without 1 M PDTC (an NF-B inhibitor) and 100 nM NF-B-p65 siRNA. Quantification of protein levels under the experimental conditions is displayed next to the blots. A representative blot is shown, and the data expressed as means SEM in each bar graph represent the average of 3 independent experiments. *, P .05 vs Ctrli; #, P .05 and ##, P .01 vs PGRN. IB, immunoblotting; IP, immunoprecipitation; pY, IRS-1 tyrosine phosphorylation; p-Akt, Akt serine 473 phosphorylation; Ctrls, controls; Veh, vehicle; Ctrli, control siRNA; NF-B p65i, NF-B p65 siRNA.

Article Snippet: Cells were transfected with a silencing RNA (small interfering RNA [siRNA]; Santa Cruz Technology) targeted for human PGRN (sc-39261), NF- B-p65 (sc-29410), and XBP-1 (sc-38627) using Lipofectamine 2000 (Invitrogen). siRNA consisting of a scrambled sequence of similar length was used as a control.

Techniques: Software, Phospho-proteomics, Expressing, Cell Culture, Western Blot, Immunoprecipitation, Control

Journal: Journal of Neuroinflammation

Article Title: Microglial progranulin differently regulates hypothalamic lysosomal function in lean and obese conditions via cleavage-dependent mechanisms

doi: 10.1186/s12974-025-03370-1

Figure Lengend Snippet: Extracellular cleavage impairs PGRN-mediated regulation of lysosomal functions in hypothalamic neurons and microglia. ( A ) Effects of treatment with PGRN alone, PGRN + NE, or PGRN + NE + SLPI on lysosomal pH (LysoSensor) and lysosomal mass (LysoTracker) in N1 hypothalamic neurons and BV2 microglia cells over a 3-h period. Approximately 100 cells were analyzed per group ( n = 5–12 wells). Scale bars, 20 μm. ( B ) DQ-BSA pulse-chase assay in N1 cells treated with PGRN alone, PGRN + NE, or PGRN + NE + SLPI. Approximately 100 cells were analyzed per group ( n = 5–8 wells). Scale bars, 25 μm. ( C ) Representative images and quantification of DQ-BSA signals in the ARH of mice fed an NCD or HFD for 10 weeks and treated with SLPI for 4 weeks prior to sacrifice. Approximately 100 cells per mouse were analyzed ( n = 3–4). Scale bars, 20 μm. ( D ) Cellular uptake and lysosomal localization of FITC-labeled PGRN and FITC-labeled PGRN fragments ( n = 100 cells). To induce PGRN cleavage, FITC-labeled PGRN was pre-incubated with NE for 1 h before treatment. Peptides were then allowed to traffic to lysosomes in N1 hypothalamic neuron cells for 1 h before fixation. The sortilin inhibitor AF38469 was used to confirm the sortilin-dependence of PGRN uptake ( n = 4–5 wells). Scale bars, 20 μm. Results are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 for comparisons between indicated groups or controls at each time point. ### p < 0.005 vs. PGRN + NE at each time point. NS, not significant

Article Snippet: FITC-conjugated PGRN, PGRN fragments (100 ng each), FITC-PGRN + sortilin inhibitor AF38469 (3 μg, MedchemExpress, #HY12802), and LysoTracker (1:2000) were then applied to the N1 cells on ice for 30 min and allowed to internalize for 5 min at 37 °C.

Techniques: Pulse Chase, Labeling, Incubation

Journal: World Journal of Gastroenterology

Article Title: Previous hepatitis B viral infection–an underestimated cause of pancreatic cancer

doi: 10.3748/wjg.v28.i33.4812

Figure Lengend Snippet: Study design and patient allocation chart. cccDNA: Covalently closed circular DNA; HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; anti-HBc: Antibody to hepatitis B core antigen; anti-HBs: Antibody to hepatitis B surface antigen; pgRNA: Pregenomic RNA.

Article Snippet: To analyze pregenomic HBV RNA (pgRNA HBV), nucleic acids were treated with RNase-free DNase I (NEB) for 30 min at 37 ºC, purified by using AmpliSens Riboprep kit (AmpliSens Biotechnologies), reverse transcribed by AmpliSens Reverta-FL (AmpliSens Biotechnologies) and quantified by AmpliSens HBV-FL (AmpliSens Biotechnologies) kit.

Techniques:

Journal: Cell Death & Disease

Article Title: Circular RNA FCHO2 promotes airway remodeling in COPD via regulating nuclear translocation of PTBP1 to repress the splicing of GRN pre-mRNA

doi: 10.1038/s41419-025-08107-9

Figure Lengend Snippet: A RIP-seq indicated that 213 mRNAs presented opposite changes in PTBP1 binding levels under circFHCO2 KD and OE conditions. RNA-seq revealed that 498 mRNAs presented opposite changes in expression levels. B Venn diagram showing 148 mRNAs whose expression levels and PTBP1 binding levels were both regulated by circFCHO2, which could be considered downstream target genes of the interaction between circFCHO2 and PTBP1. C Gene Ontology (GO) analysis revealed that 148 downstream target genes were enriched in biological processes such as positive regulation of the defense response, cell adhesion, and the inflammatory response. The positive regulation of the defense response was the most significantly enriched (lowest p value). D Heatmap indicating the PTBP1 binding level and expression level of 13 downstream target genes enriched in this biological process, and GRN mRNAs were the genes most significantly affected by circFCHO2 OE and KD among these 13 genes. E IGV visualization of the PTBP1 RIP signal on GRN pre-mRNA in circFCHO2 OE and KD BEAS-2B cells. F The levels of GRN pre-mRNA and mature GRN mRNA were quantified via RT‒PCR. The RT‒qPCR results are presented as bar plots.( n = 3) ( G ) circFCHO2 OE reduced the expression of GRN mRNA and PGRN ( n = 3). H circFCHO2 KD increased the levels of GRN mRNA and its translated protein, progranulin (PGRN). ( n = 3) ( I ) Violin plot showed that circFCHO2 OE significantly decreased PGRN secretion, whereas circFCHO2 KD increased of PGRN secretion in BEAS-2B cells by enzyme linked immunosorbent assays (ELISA).( n = 6) ( J ) PGRN protein levels were increased in the BEAS-2B cell model treated with 2% CSE. ( K , L ) Western blot ( n = 3) and immunohistochemistry (IHC)( n = 8) confirmed that PGRN protein levels were increased in COPD patients. Scale bar, 63 μm. For F , G and H , P values from two-tailed unpaired Student’s t test are shown. For I, and L, P values were calculated via Welch’s t -test. The data are shown as the means ± SDs from independent experiments.

Article Snippet: Standards, samples, reagents, and microplate preparations from Progranulin ELISA Kit (Elabscience, E-EL-H1578) were performed as described by the manufacturer.

Techniques: Binding Assay, RNA Sequencing, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Recombinant progranulin stimulates dendritic outgrowth in primary hippocampal cultures. (A) Primary hippocampal cultures were plated onto coverslips, then subjected to media changes resulting in a final concentration of 0 or 10 μg/mL recombinant human progranulin. Cultures were maintained for a total of 4 days with progranulin levels replenished in a second media change to achieve final concentrations of 0 or 10 μg/mL recombinant human progranulin. (B) Neurons treated with recombinant progranulin had greater total dendritic length after 4 days in culture than neurons not treated with recombinant progranulin (linear mixed effects model, t (217) = 2.619, p = 0.00945, n = 105–116 neurons per group from three independent cultures). Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent the median length of all neurons from each culture. Representative images of MAP2 immunostaining are shown in (C) with 50 μm scale bars.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Recombinant, Concentration Assay, Immunostaining

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Delivering progranulin to lysosomes stimulates dendritic outgrowth. (A) Primary hippocampal cultures were plated onto coverslips, then transduced with lentiviral vectors expressing GFP, progranulin (PGRN) or lysosome‐targeted progranulin (L‐PGRN) under the human PGK promoter. Cultures were maintained for 4 days before fixing and immunostaining. (B) The lenti‐PGK vectors transduced both neurons (MAP2+) and astrocytes (GFAP+), which were the two primary cell types observed in these cultures. (C) Virally expressed PGRN was secreted, while L‐PGRN exhibited no detectable secretion, even in media concentrated 5× using a centrifugal filter. (D, E) Both PGRN (linear mixed effects model, main effect of lentivirus, F (3,556) = 3.2966, p = 0.02024, PGRN versus GFP, p = 0.0404) and L‐PGRN (linear mixed effects model, p = 0.0119) stimulated dendritic outgrowth compared to GFP, while untransduced neurons exhibited comparable growth to those transduced with lenti‐GFP (linear mixed effects model, p = 0.9745). n = 124–159 neurons per group from 4 to 5 independent cultures (one culture did not include untransduced neurons). Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent the median length of all neurons from each culture. Scale bars in B and E represent 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Expressing, Immunostaining

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Delivering progranulin to neuronal lysosomes does not stimulate dendritic outgrowth. (A) Primary hippocampal cultures were plated onto coverslips and transduced with hSyn‐IRES‐GFP vectors, with an empty IRES‐GFP vector serving as a control for the PGRN‐IRES‐GFP and L‐PGRN‐IRES‐GFP vectors. Cultures were maintained for 4 days before fixing and immunostaining. (B) The lenti‐hSyn vectors selectively transduced neurons (MAP2+ cells). (C) Virally expressed PGRN was secreted, while L‐PGRN exhibited no detectable secretion, even in media concentrated 5× using a centrifugal filter. (D, E) When administered at a viral dose that gave equivalent levels of PGRN as the lenti‐PGK vector, neither PGRN nor L‐PGRN (linear mixed effects model, main effect of lentivirus, F (3,328) = 1.5083, p = 0.2122) stimulated dendritic outgrowth compared to GFP. n = 76–89 neurons per group from 3 independent cultures. (F, G) When administered at a dose of equivalent viral copies, which produced higher levels of PGRN than the lenti‐PGK vector, PGRN stimulated dendritic outgrowth compared to GFP (linear mixed effects model, main effect of lentivirus, F (2,606) = 3.3301, p = 0.03644, PGRN vs. Ctrl, p = 0.0113), while L‐PGRN did not (linear mixed effects model, p = 0.3766). n = 202–207 neurons per group from 4 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent median length of all neurons from each culture. Scale bars in B, E, and G represent 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Plasmid Preparation, Control, Immunostaining, Produced

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Delivering progranulin to astrocytic lysosomes stimulates dendritic outgrowth. (A) Primary hippocampal cultures were plated onto coverslips and transduced with GFAP‐IRES‐GFP vectors, with the empty vector serving as a control for the PGRN‐IRES‐GFP and L‐PGRN‐IRES‐GFP vectors. Cultures were maintained for 4 days before fixing and immunostaining. (B) Lenti‐GFAP vectors selectively transduced astrocytes (GFAP+ cells). (C) Virally expressed PGRN was secreted, while L‐PGRN exhibited no detectable secretion, even in media concentrated 5× using a centrifugal filter. (D, E) Both PGRN (linear mixed effects model, main effect of lentivirus, F (4,801) = 5.4101, p = 0.0002662, PGRN vs. Ctrl, p < 0.0001) and L‐PGRN (linear mixed effects model, L‐PGRN vs. Ctrl, p = 0.00155) stimulated dendritic outgrowth compared to the control vector, while untransduced neurons did not have significantly different growth from those transduced with lenti‐IRES‐GFP (linear mixed effects model, p = 0.1539). As an additional control, this experiment included neurons transduced with lysosome‐targeted GFP (L‐GFP), which also did not stimulate growth relative to the control vector (linear mixed effects model, p = 0.1443). n = 139–193 neurons per group from 3 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent the median length of all neurons from each culture. Scale bars in B and E represent 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Plasmid Preparation, Control, Immunostaining

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Enhanced dendritic outgrowth in neurons co‐cultured with astrocytes transduced with L‐PGRN. (A) mature hippocampal astrocyte cultures were obtained by plating in DMEM with 10% serum and culturing for 1 week, then passaging to transwell inserts in serum‐free medium and transducing with lenti‐PGK vectors 4 days later. These transwell inserts containing mature astrocytes were placed over new primary hippocampal cultures, which were harvested after 4 days for immunostaining. Representative progranulin immunostaining in astrocytes is shown in (B). (C) PGRN was secreted by astrocytic cultures, while L‐PGRN was not. (D) Lack of progranulin secretion was confirmed by ELISA of conditioned media of several astrocyte cultures. Media from cultures transduced with lenti‐PGK‐GFP exhibited low background signal for human progranulin (hPGRN), which was dramatically increased in cultures transduced with lenti‐PGK‐PGRN (ANOVA main effect of lentivirus, F (2,12) = 6.974, p = 0.0098, PGRN versus GFP p = 0.0123 and PGRN vs. L‐PGRN p = 0.0297 by Tukey's post hoc test, n = 16 wells from 5 independent cultures). Cultures transduced with lenti‐PGK‐L‐PGRN did not differ from GFP ( p = 0.877 by Tukey's post hoc test) and also had dramatically less hPGRN in conditioned media than cultures transduced with lenti‐PGK‐PGRN ( p = 0.0297 by Tukey's post hoc test). (E) L‐PGRN was delivered to lysosomes of primary astrocytes, as shown by colocalization of the HA tag from virally expressed L‐PGRN with the lysosomal protease cathepsin D (CatD). (F, G) Despite the lack of progranulin secretion, neurons co‐cultured with astrocytes transduced with L‐PGRN had greater dendritic outgrowth compared to neurons cultured with GFP‐treated astrocytes (linear mixed effects model, main effect of lentivirus, F (2,580) = 7.6264, p = 0.0005379, L‐PGRN vs. GFP, p = 0.000619) while neurons co‐cultured with untransduced astrocytes had similar dendritic outgrowth as neurons cultured with GFP‐treated astrocytes (linear mixed effects model, p = 0.876719). n = 180–206 neurons per group from 4 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent median length of all neurons from each culture. Scale bars represent 50 μm in B and G and 10 μm in E. Panel A created at Biorender.com , Arrant (2025) https://BioRender.com/cme038k .

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Cell Culture, Transduction, Passaging, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: L‐PGRN reduces transcriptomic signatures of cellular reactivity in primary astrocytes. (A) To better understand how delivering progranulin to lysosomes changes the phenotype of primary astrocytes, we cultured mature astrocytes using the same procedure as previously used for neuronal co‐cultures. Instead of co‐culturing with neurons, we switched astrocytes to a “co‐culture medium” identical to that used in co‐cultures. After 4 days in co‐culture medium, we harvested RNA from these astrocytes and conducted bulk RNA sequencing. (B) Nearly all cells in these cultures were immunoreactive for the astrocytic markers GFAP and S100β, and (C) bulk RNA sequencing confirmed the enrichment of astrocyte marker genes versus other cell types. (D) Gene set enrichment analysis of sequencing data using Hallmark pathways suggested potential increases in proliferation and decreases in cellular reactivity. n = 4 samples per treatment from 4 independent cultures. Panel A created at Biorender.com , Arrant (2025) https://BioRender.com/560o6wr . Raw counts of genes in panel C are provided in Table , and a full list of enriched Hallmark pathways from panel D is provided in Table .

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Cell Culture, Co-Culture Assay, RNA Sequencing, Marker, Sequencing

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Reducing the number of astrocytes occludes the pro‐growth effects of L‐PGRN. (A) Primary hippocampal cultures were plated onto coverslips, transduced with lenti‐PGK‐GFP or L‐PGRN, then treated with 5 μM Ara‐C approximately 24 h after plating. Cultures were maintained for a total of 4 days. (B) Ara‐C treatment modestly reduced the number of neurons in these cultures (linear mixed effects model main effect of Ara‐C, F (1,108) = 16.5707, p < 0.0001, ** = p = 0.00122 for L‐PGRN vs. L‐PGRN+Ara‐C by Tukey's post hoc test). L‐PGRN–treated cultures treated with Ara‐C also had slightly fewer neurons per visual field than GFP‐treated cultures treated with Ara‐C (linear mixed effects model main effect of lentivirus, F (1,108) = 6.3728, p = 0.01304, * = p = 0.04435 for GFP + Ara‐C vs. L‐PGRN+Ara‐C by Tukey's post hoc test), though there was not a significant difference between lentiviral groups under control conditions ( p = 0.65663 by Tukey's post hoc test). (C) Ara‐C nearly completely eliminated astrocytes from these cultures (linear mixed effects model main effect of Ara‐C, F (1,108) = 74.08, p < 0.0001, *** = p < 0.001 for GFP vs. GFP + Ara‐C and L‐PGRN vs. L‐PGRN+Ara‐C by Tukey's post hoc test). There was not a significant difference between lentiviral treatment groups in the number of astrocytes per visual field (linear mixed effects model main effect of lentivirus, F (1,108) = 0.707, p = 0.4023). n = 28–29 coverslips from four independent cultures. Violin and box plots represent the distribution of cell count for all coverslips analyzed, and dots represent median cell count from each culture. (D, E) L‐PGRN+Ara‐C wells expressed less progranulin than L‐PGRN + vehicle wells by immunoblot (paired t ‐test, t (2) = 4.403, p = 0.0479, n = 10–11 replicates from 3 independent cultures). (F, G) However, analysis of progranulin immunoreactivity in neurons revealed a non‐significant trend for lower progranulin (paired t test, t (3) = 2.237, p = 0.1113, n = 12 replicates from 4 independent cultures). Scale bars in G represent 100 μm. (H) Analysis of dendritic length in vehicle‐treated cultures replicated our prior finding that L‐PGRN promotes dendritic outgrowth (linear mixed effects model, main effect of lentivirus, F (1,996) = 5.1756, p = 0.02312, ** = p = 0.00802 for GFP vs. L‐PGRN by Tukey's post hoc test), but also revealed that Ara‐C treatment stimulated dendritic outgrowth (linear mixed effects model main effect of Ara‐C, F (1,996) = 15.5234, p < 0.0001, *** = p < 0.001 for GFP vs. GFP + Ara‐C by Tukey's post hoc test) and that among Ara‐C treated neurons, those transduced with L‐PGRN did not have significantly different dendritic growth from those transduced with GFP (linear mixed effects model, p = 0.99088). n = 215–275 neurons per group from 4 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent median length of all neurons from each culture. Representative images of MAP2 immunostaining are shown in (I) with 50 μm scale bars.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Control, Cell Counting, Western Blot, Immunostaining

Journal: Nucleic Acids Research

Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

doi: 10.1093/nar/gku836

Figure Lengend Snippet: Activation of the mouse Oct4 and Nanog loci by TALE-As and dCas9-As/gRNAs. ( A ) Schematic diagram of the dCas9-As evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-As. gRNA expression is controlled by U6 promoter, and EF1a-mCherry is used to detect the integration of the vector into the genome. A blasticidin resistance and the reverse tetracycline transactivator (rtTA) cassettes were also linked to the mCherry cassette by T2A peptides. PB-5TR and PB-3TR are the two terminal repeat sequences of the piggyBac (PB) transposon. ( B ) Schematic diagram of the TALE and dCas9/gRNA targeting sites at the mouse Oct4 and Nanog enhancers. Red arrows indicate the gRNA targeting sites and the blue arrows mark the TALE sites. ( C ) Activation of the Oct4 distal enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( D ) qRT-PCR analysis of the Oct4 mRNA levels in MEFs expressing the TALE-As or dCas9-As/gRNAs. ( E ) Activation of the Nanog enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( F ) qRT-PCR analysis of the Nanog mRNA levels in EpiSCs expressing the TALE-As or dCas9-As/gRNAs. All gene expression values were normalized to Gapdh . Results were representative of three independent experiments and were presented as ±SD, n = 3. * P < 0.05.

Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Gene Expression

Journal: Nucleic Acids Research

Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

doi: 10.1093/nar/gku836

Figure Lengend Snippet: Repression of the Oct4 and Nanog loci by TALE-Rs and dCas9-Rs/gRNAs. ( A ) Schematic diagram of the dCas9-R designs evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-R. The gRNA vector used was the same as described in Figure . ( B ) Repression of the endogenous Oct4 locus in Oct4-GFP ES cells indicated by the reduction of GFP intensity in flow cytometric analysis on day 0 and day 3 of expression of the TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–4 of the Oct4 distal enhancer (Gated mCherry + for TALEs and mCherry + /BFP + for dCas9-As/gRNAs). ( C ) Comparison of Oct4 expression levels in Oct4-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Site 1 or Site 3 of the Oct4 distal enhancer by qRT-PCR. ( D ) The repressive effect of TALE-R and dCas9-Rs/gRNAs targeting at the Site 3 of the Oct4 distal enhancer on MEF reprogramming. MEFs were reprogrammed with Dox inducible CKS and Lrh1 (CKSL) factors together with the TALE-R or dCas9-R/gRNAs as in (C). ‘CKSL+’ indicates that all transfections have CKSL. ‘−’ is the CKSL only control (no repressor). ‘CKS only’ is the reprogramming negative control. ( E ) Reprogramming using FACS-sorted MEFs (as described in 2E) to control transfection variability. Wild-type MEFs were transfected and sorted on day 2 for GFP + /mCherry + in the TALE-R transfection and for GFP + /mCherry + /BFP + in dCas9-R/gRNAs (PL-R) experiments. Both TALE-Rs and dCas9-R/gRNAs (PL-R) targeted the Site 2–4 of the Oct4 distal enhancer. Sorted MEFs were re-plated (20 000 cells/ well) for reprogramming and iPSC colonies were scored by AP staining. ( F ) The repressive effect of TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–2 of the Nanog 5 kb enhancer in Nanog-GFP ES cells. Site 1 is located outside the enhancer region whereas Site 2 is inside. The Nanog repression was demonstrated by the increase of the GFP low/dim fraction in Nanog-GFP ES cells. ( G ) Endogenous N anog mRNA levels in Nanog-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Sites 1–2. ( H ) Repression of Klf4- mediated EpiSC reprogramming to iPSCs by the TALE-R and dCas9-Rs targeting at the Site 2 of the Nanog 5 kb enhancer. ‘Klf4+’ refers to the transfections combined with Klf4 and ‘−’ is the Klf4 control (no effector). Results were representative of three independent experiments and were presented as ±SD, n = 3.

Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

Techniques: Expressing, Plasmid Preparation, Comparison, Quantitative RT-PCR, Transfection, Control, Negative Control, Staining