Journal: Frontiers in Immunology

Article Title: Co-Expression of the B-Cell Key Transcription Factors Blimp-1 and IRF4 Identifies Plasma Cells in the Pig

doi: 10.3389/fimmu.2022.854257

Figure Lengend Snippet: Antibodies and reagents used for FCM analysis.

Article Snippet: Freshly isolated PBMCs were resuspended in RPMI 1640 with stable glutamine (PAN-Biotech), supplemented with 5% (v/v) heat-inactivated FCS (Gibco™, Thermo Fisher Scientific), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (PAN-Biotech) and incubated with mAbs against CD3 (clone PPT3, mouse IgG1 isotype, in-house) and CD16 (clone G7, mouse IgG1 isotype, Bio-Rad, Hercules, CA, USA) on ice for 20 min.

Techniques: Labeling, Ex Vivo

Journal: Frontiers in Immunology

Article Title: Co-Expression of the B-Cell Key Transcription Factors Blimp-1 and IRF4 Identifies Plasma Cells in the Pig

doi: 10.3389/fimmu.2022.854257

Figure Lengend Snippet: Frequencies of Ig-classes within Blimp-1 + IRF4 + PCs at different anatomical locations. (A) Live lymphocytes were gated on CD79α + IRF4 high cells and further into Blimp1 + IRF4 + PCs (red). Gray color represents the CD79α + IRF4 dim population. (B) Frequencies for IgM, IgG, and IgA expressing cells were investigated in blood, spleen, mediastinal lymph node (Ln Med), mesenteric lymph node (Ln Mes), bone marrow (BM), and lung within Blimp-1 + IRF4 + cells. Pseudocolor plots of the six anatomical locations are shown for one representative animal and frequencies of positive cells are indicated in the quadrants. (C) The graph on the left shows the frequencies of Blimp-1 + IRF4 + PCs within live lymphocytes of all animals and organs analyzed. The following three graphs display the frequencies of IgM + , IgG + , and IgA + cells within Blimp-1 + IRF4 + PCs (n = 6 for all organs except Ln Med with n = 5), horizontal bars in the graphs indicate respective mean values.

Article Snippet: Freshly isolated PBMCs were resuspended in RPMI 1640 with stable glutamine (PAN-Biotech), supplemented with 5% (v/v) heat-inactivated FCS (Gibco™, Thermo Fisher Scientific), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (PAN-Biotech) and incubated with mAbs against CD3 (clone PPT3, mouse IgG1 isotype, in-house) and CD16 (clone G7, mouse IgG1 isotype, Bio-Rad, Hercules, CA, USA) on ice for 20 min.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Co-Expression of the B-Cell Key Transcription Factors Blimp-1 and IRF4 Identifies Plasma Cells in the Pig

doi: 10.3389/fimmu.2022.854257

Figure Lengend Snippet: FACS sorting of live PCs and functional analysis. (A) CD3/CD16 depleted blood-derived cells were further gated for doublet discrimination (FSC-A vs. FSC-H) and exclusion for CD172a as well as CD3 + CD16 + cells remaining from the MACS sort. CD3 - CD16 - CD172a - cells were finally sorted based on their expression of CD49d and FSC-A properties into two populations: CD49d -/+ (blue) and CD49d high FSC-A high (red). (B) After sorting, cell populations were stained for CD79α, Blimp-1, and IRF4. Stacked histograms represent the expression of CD79α in both sorted populations as well as in the corresponding unstained controls (gray). The pseudocolor plot shows the frequency of Blimp-1 + IRF4 + cells within the CD49d high sorted population. Percentages of Blimp-1 + IRF4 + cells in the two sorted populations are summarized in the graph on the right (n = 4). (C) Two representative wells of the B-cell IgG ELISpot for the sorted populations are shown on the left. The numbers of counted spots for all animals analyzed are summarized in the graph on the left and indicated within 2 × 10 4 seeded cells (n = 4). The graph in the middle shows the frequencies of IgG-producing cells calculated within total Blimp-1 + IRF4 + cells for the CD49d high -sorted population of the respective animals (n = 4). The graph on the right shows frequencies of IgG-producing cells calculated within total IgG + Blimp-1 + IRF4 + cells as analyzed in parallel according to Figure 3 in three of the four animals. Horizontal bars represent the respective mean values.

Article Snippet: Freshly isolated PBMCs were resuspended in RPMI 1640 with stable glutamine (PAN-Biotech), supplemented with 5% (v/v) heat-inactivated FCS (Gibco™, Thermo Fisher Scientific), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (PAN-Biotech) and incubated with mAbs against CD3 (clone PPT3, mouse IgG1 isotype, in-house) and CD16 (clone G7, mouse IgG1 isotype, Bio-Rad, Hercules, CA, USA) on ice for 20 min.

Techniques: Functional Assay, Derivative Assay, Expressing, Staining, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines

doi: 10.1038/s41598-025-11124-5

Figure Lengend Snippet: ARL13B -KO RPE1 cells have no cilia. ( a ) A schematic diagram of CRISPR/Cas9 strategy to make frameshift mutations in human ARL13B gene. The scheme of the exon organization of the human ARL13B gene is illustrated at the top. The middle shows parts of the WT genomic sequences of exon 1 and exon 5, where sequences of two sgRNAs, sg1 and sg2, are highlighted in light blue. Underlined sequences are protospacer-adjacent motifs or PAMs. The bottom shows genomic sequences at sgRNA target sites of two alleles in sg1#a and sg2#4 cell lines. Sequences are aligned with the corresponding WT ones. Inserted and deleted bases are colored red and indicated by “−”, respectively. “+2” and “− 7” indicate insertion of 2 and deletion of 7 bases, respectively. ( b ) Western blotting of endogenous ARL13B in parental and KO RPE1 cells. Cell lysates from indicated cell lines were subjected to 12% SDS-PAGE followed by immunoblotting using anti-ARL13B and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (loading control) antibodies. Molecular weight markers are labeled to the right of the immunoblot. ( c ) Fluorescence imaging of endogenous ARL13B and acetylated tubulin in parental and KO RPE1 cells. Indicated cells were fixed and co-immunostained by antibodies against indicated cilium markers before being subjected to wide-field microscopy. ( d ) Quantification of the ciliogenesis percentage in parental and KO RPE1 cells. Images acquired as described in ( c ) were analyzed. n = 3 independent experiments with ≥ 100 cells analyzed in each case. ( e ) Fluorescence imaging of endogenous glutamylated tubulin and pericentrin in parental and KO RPE1 cells, similar to ( c ). ( f ) Silent mutations in ARL13B R -GFP make it resistant to sgRNAs. Regions of the ARL13B R coding sequence, corresponding to sg1, sg2, sg4, and sg5 (colored blue), are shown with introduced multiple silent mutations colored red. Protospacer-adjacent motifs, or PAMs, are underlined. Superscript numbers indicate base pair positions in the coding sequence. ( g ) ARL13B R -GFP can rescue the no-cilium phenotype of ARL13B -KO cell lines. Parental RPE1, sg1#a, and sg2#4 cells expressing ARL13B R -GFP were processed for immunofluorescence to label endogenous acetylated tubulin and pericentrin. Arrows indicate ciliated cells. In ( c , e , and g ), the boxed region is enlarged at the lower left corner, and the scale bar represents 10 μm. ( h–j ) ARL13B R -GFP expressing sg1#a cells exhibit normal cilia. Images acquired as described in ( g ) were used for quantification. Cilia were identified by acetylated tubulin. Panel h shows the ciliogenesis percentage in GFP-positive parental and sg1#a cells (indicated by “+”). It also displays the ciliogenesis percentage of GFP-negative sg1#a cells from the same coverslips (indicated by “−”). Quantification was from n = 3 independent experiments with ≥ 100 cells analyzed in each experiment. In (i), the cilium length was measured in ciliated GFP-positive cells. Quantification was from n = 3 independent experiments with ≥ 30 cells analyzed in each experiment. In ( j ), the CPIR of ARL13B R -GFP was quantified in ciliated GFP-positive cells. Quantification was from n = 3 independent experiments with ≥ 30 cells analyzed in each experiment. In ( d , h – j ), the error bar represents the mean ± standard deviation .

Article Snippet: Rabbit polyclonal antibodies (pAbs) against ARL13B (in-house made; 1:1000 for IF) , ARL13B (ProteinTech, #17711-AP, 1:1000 for IF and 1:3000 for WB), and pericentrin (Abcam, #ab4448, 1:1000 for IF).

Techniques: CRISPR, Genomic Sequencing, Western Blot, SDS Page, Control, Molecular Weight, Labeling, Fluorescence, Imaging, Microscopy, Sequencing, Expressing, Immunofluorescence, Standard Deviation

Journal: Scientific Reports

Article Title: Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines

doi: 10.1038/s41598-025-11124-5

Figure Lengend Snippet: ARL13B -KO RPE1 cells prepared using previously reported sgRNAs have no cilia either. ( a ) The genotypes of ARL13B -KO RPE1 cell lines, sg4#2 and sg5#3, using previously published sgRNAs. See the legend of Fig. a. ( b – c ) Endogenous ARL13B is undetectable by immunofluorescence imaging and no cilia could be found in sg4#2 and sg5#3 cells. See the legend of Fig. c, e ( d ) Exogenously expressed ARL13B R -GFP can rescue the no-cilium phenotype of sg4#2 and sg5#3 cells. See the legend of Fig. g. In ( b – d ), the boxed region is enlarged at the lower left corner, and the scale bar represents 10 μm.

Article Snippet: Rabbit polyclonal antibodies (pAbs) against ARL13B (in-house made; 1:1000 for IF) , ARL13B (ProteinTech, #17711-AP, 1:1000 for IF and 1:3000 for WB), and pericentrin (Abcam, #ab4448, 1:1000 for IF).

Techniques: Immunofluorescence, Imaging

Journal: Scientific Reports

Article Title: Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines

doi: 10.1038/s41598-025-11124-5

Figure Lengend Snippet: Multiple ARL13B regions or domains are required for ciliogenesis. ( a ) The schematic diagram illustrating the organization of WT and mutant ARL13B, which were constructed in the background of ARL13B R -GFP. The top shows the WT ARL13B. Different functional regions and domains are indicated with AA positions. Interacting proteins previously reported are labeled above the corresponding regions or domains and shaded green. The AA sequences of PAL and RVEP motifs are shown below. ARL13B mutants with point mutations or truncations are illustrated below the WT. On the right is the qualitative summary of the cilium localization of corresponding GFP-tagged WT and mutant ARL13B (see below for details). “+” indicates a positive cilium localization; “−” indicates a negative cilium localization or no ciliogenesis; “W” indicates a weak cilium localization. ( b ) The cellular distribution of GFP-tagged WT and mutant ARL13B R -GFP. Parental and sg1#a RPE1 cells expressing GFP-tagged WT and mutant ARL13B R -GFP were immunostained for indicated proteins. The boxed region is enlarged at the lower left corner, and the scale bar represents 10 μm. ( c ) The cilium localization percentage (Parental RPE1 cells) and cilium biogenesis percentage (sg1#a RPE1 cells) of GFP-positive cells. The images acquired as described in ( b ) were analyzed. n = 3 independent experiments with ≥ 100 cells analyzed in each. ( d – f ) The RVEP motif is non-essential for ciliogenesis but essential for the cilium length and the cilium enrichment of ARL13B R -GFP in ARL13B -KO cells. Images acquired as described in ( b ) were quantified. GFP-positive cells were analyzed for the ciliogenesis percentage ( d ), the cilium length ( e ), and the CPIR ( f ) of ARL13B R -GFP. See legend of Fig. h–j. p values are from unpaired, two-tailed t -tests. Not significant or NS, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005. In ( c , d – f ), the error bar represents the mean ± standard deviation.

Article Snippet: Rabbit polyclonal antibodies (pAbs) against ARL13B (in-house made; 1:1000 for IF) , ARL13B (ProteinTech, #17711-AP, 1:1000 for IF and 1:3000 for WB), and pericentrin (Abcam, #ab4448, 1:1000 for IF).

Techniques: Mutagenesis, Construct, Functional Assay, Labeling, Expressing, Two Tailed Test, Standard Deviation

Journal: Scientific Reports

Article Title: Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines

doi: 10.1038/s41598-025-11124-5

Figure Lengend Snippet: Joubert syndrome mutation R200C, but not R79Q and Y86C, is essential for the ciliogenesis function of ARL13B. ( a ) The schematic diagram illustrates the organization of ARL13B and its three Joubert syndrome mutants. See the legend of Fig. a. ( b – c ) The cellular distribution, cilium localization, and ciliogenesis percentage of GFP-tagged Joubert syndrome mutant ARL13B R . The boxed region is enlarged at the lower left corner, and the scale bar represents 10 μm. See the legend of Fig. b, c. ( d ) The images acquired as described in ( b – c ) were analyzed. n = 3 independent experiments with ≥ 30 cells analyzed in each. The error bar represents the mean ± standard deviation.

Article Snippet: Rabbit polyclonal antibodies (pAbs) against ARL13B (in-house made; 1:1000 for IF) , ARL13B (ProteinTech, #17711-AP, 1:1000 for IF and 1:3000 for WB), and pericentrin (Abcam, #ab4448, 1:1000 for IF).

Techniques: Mutagenesis, Standard Deviation

Journal: Scientific Reports

Article Title: Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines

doi: 10.1038/s41598-025-11124-5

Figure Lengend Snippet: Hedgehog signaling is abolished in ARL13B -KO cells. ( a ) SAG treatment increased the cilium localization of SMO in RPE1 cells. RPE1 cells treated with or without (control) 0.5 μM SAG for 24 h were fixed and processed for immunofluorescence labeling of endogenous SMO and ARL13B. Arrows and arrowheads indicate SMO-positive and -negative cilia, respectively. ( b ) Percentage of ciliated cells with SMO-positive cilia. Images acquired as described in ( a ) were analyzed. n = 4 independent experiments. ( c ) Normalized transcription levels of SMO , PTCH1 , and GLI1 genes in parental RPE1 or sg1#a cells with or without (control) 0.5 μM SAG treatment for 24 h. The transcription was quantified by qPCR and normalized by the corresponding control. n = 5 independent experiments. ( d – e ) Exogenously expressed ARL13B R -GFP can rescue the SAG-induced cilium localization of SMO in ARL13B -KO cells. Parental RPE1 or sg1#a cells expressing ARL13B R -GFP were treated with or without (control) 0.5 μM SAG for 24 h. Cells were subsequently fixed and processed for immunofluorescence labeling of endogenous SMO and pericentrin ( d ). Arrows indicate ciliated cells. The percentage of ARL13B R -GFP expressing ciliated sg1#a cells with SMO-positive cilia is quantified in ( e ). n = 3 independent experiments with ≥ 100 cells analyzed in each. In ( a , d ), scale bar, 10 µm. In ( b , c , e ), the error bar represents the mean ± standard deviation, and p values were from unpaired, two-tailed t -tests. NS, p > 0.05; ∗, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005.

Article Snippet: Rabbit polyclonal antibodies (pAbs) against ARL13B (in-house made; 1:1000 for IF) , ARL13B (ProteinTech, #17711-AP, 1:1000 for IF and 1:3000 for WB), and pericentrin (Abcam, #ab4448, 1:1000 for IF).

Techniques: Control, Immunofluorescence, Labeling, Expressing, Standard Deviation, Two Tailed Test