Journal: bioRxiv

Article Title: The primary cilium controls programmed cell death via its proteasome-regulating function

doi: 10.64898/2026.01.07.698216

Figure Lengend Snippet: ( A ) Coimmunoprecipitation in NIH3T3 cells. MYC-tagged MOAP1 full-length protein and FLAG-tagged RID (domain of RPGRIP1L) were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. IP, immunoprecipitation; WB, western blot. ( B ) In situ proximity ligation assay ( in situ PLA) on serum-starved NIH3T3 cells. Cell nuclei are stained in blue by DAPI and cilia are marked by the product of a transiently transfected IFT88-EYFP construct. The PLA+ signal is shown in red. The scale bar (in white) represents a length of 2 μm. ( C ) Immunofluorescence on serum-starved NIH3T3 cells. Super-resolution image was acquired by using Airyscan microscopy. The ciliary axoneme is labelled in green by detyrosinated α-tubulin, the basal body is marked in blue by γ-tubulin. MOAP1 is shown in red. The scale bar (in white) represents a length of 5 μm. ( D ) Immunofluorescence on serum-starved NIH3T3 cells. Super-resolution image was acquired by using dSTORM. The ciliary axoneme is labelled in green by detyrosinated α-tubulin, the basal body is marked in turquoise by γ-tubulin. MOAP1 is shown in magenta. The scale bar (in white) represents a length of 1 μm. The depicted cilium is shown in three different modes of representation – the point cloud mode (on the left), the cluster mode (in the middle) and point splatting mode (on the right). In the point splatting mode, the yellow arrow points to the portion of MOAP1 that protrudes into the basal body up to the transition zone (shown in white). ( E ) Immunofluorescence on serum-starved NIH3T3 cells. The ciliary axoneme is stained in green by detyrosinated α-tubulin and the BB in blue by γ-tubulin. The scale bar (in white) represents a length of 1 μm. 20 cilia per genotype were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + DMSO: **** P <0.0001; Rpgrip1l -/- vs. Rpgrip1l -/- + SFN: **** P <0.0001; Rpgrip1l -/- + DMSO vs. Rpgrip1l -/- + SFN: **** P <0.0001). ns = not significant. ( F ) Immunofluorescence on serum-starved NIH3T3 cells. The ciliary axoneme is stained in green by detyrosinated α-tubulin and the BB in blue by γ-tubulin. The scale bar (in white) represents a length of 1 μm. 20 cilia per genotype were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + siRNA Ctrl.: **** P = 0.0009; Rpgrip1l -/- vs. Rpgrip1l -/- + siRNA Moap1 : **** P <0.0001; Rpgrip1l - /- siRNA Ctrl. vs. Rpgrip1l -/- + siRNA Moap1 : **** P <0.0001). ns = not significant; Ctrl., control. ( G ) Immunofluorescence on serum-starved NIH3T3 cells. Cell nuclei are stained in blue by DAPI. Cells undergoing PCD are marked in red by CC3. The scale bar (in white) represents a length of 10 μm. At least 800 cells per genotype and treatment were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + siRNA Ctrl.: *** P = 0.0009; Rpgrip1l -/- vs. Rpgrip1l - /- + siRNA Moap1 : **** P <0.0001; Rpgrip1l -/- + siRNA Ctrl. vs. Rpgrip1l -/- + siRNA Moap1 : ** P = 0.0013). ns = not significant; Ctrl., control.

Article Snippet: Mouse full-length MOAP1 was cloned into the MYC-tagged pCMV vector (Clontech).

Techniques: Immunoprecipitation, Western Blot, In Situ, Proximity Ligation Assay, Staining, Transfection, Construct, Immunofluorescence, Microscopy, Control

Journal: Human Genetics

Article Title: Distinct regulatory elements of SLC6A14 expression contribute to modification of cystic fibrosis phenotypes

doi: 10.1007/s00439-025-02800-7

Figure Lengend Snippet: Distal enhancer activity with augmented response to IRF1 depending on rs4446858 SNP allele. A Juxtaposition of the 2.1 kb segment of the 17 kb sub-region in forward (F) or reverse (R) orientation to the core promoter increased reporter gene activity in MOR cells, but not in CFPAC-1 cells. Luciferase activity values were determined as in Fig. , except that activity of core promoter only was set as 1 for comparisons. Two independent experiments were performed, each with two to four technical replicates for each vector. B The 2.1 kb segment with the most common SNP combination (haplotype D1; black), with alternate alleles A and C for rs12009976 and rs4446858, respectively (haplotype D4; pale) and with the alternate C allele for rs4446858 only (grey) yielded comparable reporter gene activities. Each bar represents the mean of five to ten replicates from two independent experiments. C Inclusion of exogenous IRF1 by co-transfection revealed further enhancement of the reporter gene activity with the alternate C allele of rs4446858. The exogenous inclusion of IRF2 modestly reduced enhancer activity, compared to the pCMV control vector only, and was independent of the C allele. A combination of IRF2 with IRF1 reduced the effect of IRF1 alone. The reporter gene assays were performed as in ( A ) except the core promoter fragment with pGL4.10 plus pCMV was set as 1 for comparisons. Each bar represents mean of six to twelve replicates from two independent experiments. For all experiments error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 using unpaired t tests.

Article Snippet: The SLC6A14 insert was ligated to the EcoRI/BamHI-digested pTetOne vector (Clontech, 634310), and IRF1 , IRF2 inserts were ligated to the NotI-digested pCMV vector (Clontech, 631719), replacing LacZ .

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Cotransfection, Control