🩸Today's question! See the answer and explanation on the next slide.
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![Microtubule mass is reduced in SDS patient cells. (A) mRNA levels of MAP7D1 in control and patient fibroblasts. The experiment was performed in three biological replicates, each including three independent technical replicates. qRT-PCR data in A represent the MAP7D1/ACTB values obtained by normalizing MAP7D1 mRNA expression levels in each experimental group to its respective ACTB mRNA expression level using the ΔΔCT method and compared with those of the control experiment group. (B) Quantification of endogenous MAP7D1 protein levels in control and patient fibroblasts. The expression level of endogenous MAP7D1 protein was analyzed by western blotting (left) with β-actin as an internal loading control. The experiment was performed in three biological replicates. Quantification of band densities is represented in the bar graph (right). Data were normalized to those of β-actin and compared with those of the control experiment group. (C) Confocal images showing colocalization of MAP7D1 protein with microtubules in control and patient fibroblasts (left and right panel, respectively) using immunocytochemistry. Antibody labeling for MAP7D1 is shown red, microtubules (stained for β-tubulin) are shown in green. Shown are representative of three biological replicates including two independent technical replicates. Merged images are shown on the right of each panel. Scale bars: 20 µm. (D) Quantification of the cell area in control and patient fibroblasts is represented in the bar graph (n≥45). (E) Quantification of the fluorescence intensity [measured as active fluorescent unit (AFU)] of microtubules in control and patient fibroblasts is represented in the bar graph (n≥50). t-test was used for all statistical analysis. Error bars represent the mean±s.e.m. *P<0.05, ***P<0.001, ****P<0.0001.](https://cdn.bsky.app/img/feed_thumbnail/plain/did:plc:5jupfg23qpjk2643o6aeftly/bafkreidk3z2fsmgpdnf6upqx23k2pbvvpf7xwtllbjvua7d43jg2xb3r3q)
Microtubule mass is reduced in SDS patient cells. (A) mRNA levels of MAP7D1 in control and patient fibroblasts. The experiment was performed in three biological replicates, each including three independent technical replicates. qRT-PCR data in A represent the MAP7D1/ACTB values obtained by normalizing MAP7D1 mRNA expression levels in each experimental group to its respective ACTB mRNA expression level using the ΔΔCT method and compared with those of the control experiment group. (B) Quantification of endogenous MAP7D1 protein levels in control and patient fibroblasts. The expression level of endogenous MAP7D1 protein was analyzed by western blotting (left) with β-actin as an internal loading control. The experiment was performed in three biological replicates. Quantification of band densities is represented in the bar graph (right). Data were normalized to those of β-actin and compared with those of the control experiment group. (C) Confocal images showing colocalization of MAP7D1 protein with microtubules in control and patient fibroblasts (left and right panel, respectively) using immunocytochemistry. Antibody labeling for MAP7D1 is shown red, microtubules (stained for β-tubulin) are shown in green. Shown are representative of three biological replicates including two independent technical replicates. Merged images are shown on the right of each panel. Scale bars: 20 µm. (D) Quantification of the cell area in control and patient fibroblasts is represented in the bar graph (n≥45). (E) Quantification of the fluorescence intensity [measured as active fluorescent unit (AFU)] of microtubules in control and patient fibroblasts is represented in the bar graph (n≥50). t-test was used for all statistical analysis. Error bars represent the mean±s.e.m. *P<0.05, ***P<0.001, ****P<0.0001.
Seren Kucukvardar & Arzu Karabay, for the first time, functionally identify microtubule-associated MAP7D1 as a novel causative gene for Shwachman−Diamond syndrome using patient fibroblasts and show cell cycle-related roles of MAP7D1 protein. doi.org/10.1242/dmm....
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