stealth control Search Results


control stealth rna  (Oligos Etc)
90
Oligos Etc control stealth rna
CCDC28B interacts with the kinesin 1 components <t>KLC1</t> and KIF5B. ( A ) The co-expression of CCDC28B and KLC1 allows cdc25H yeast cells to grow at the non-permissive temperature of 37 °C only on galactose that triggers expression from the pMyr construct. Controls: MAFB-MAFB (positive); MAFB-Lamin C (negative), ColI-MAFB (negative), pSOS EV-pMyr EV (negative) and pSOS CCDC28B-pMyr EV (negative). ( B ) HA-CCDC28B is detected only in the Myc-KLC1 immunoprecipitate. Cell lysates are shown to control for protein input. Bands were cropped from the same blot which is shown in Fig. . ( C ) Immunoprecipitation of overexpressed CCDC28B in Hek293 cells with the specific single domain llama antibody (VHH) results in the co-immunoprecipitation of additional proteins. Arrows indicate gel bands analyzed by mass spectrometry. An irrelevant VHH was used as control. The gel was cut in two to avoid saturation of the VHH bands when silver-staining the upper part of the gel. ( D ) The VHH against CCDC28B was used for immunoprecipitation and specific antibodies were used to detect KIF5B, KLC1, α-tubulin and CCDC28B. Cell lysates show the corresponding proteins in the extracts used for immunoprecipitation. Bands shown were cropped from the original blot. The full-length membrane was cut and exposed to the different antibodies (see Fig. for blots and for details).
Control Stealth Rna, supplied by Oligos Etc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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chemically modified stealth small interfering rnas (sirnas) targeting fbxo31 and control sirna  (Ribobio co)
90
Ribobio co chemically modified stealth small interfering rnas (sirnas) targeting fbxo31 and control sirna
CCDC28B interacts with the kinesin 1 components <t>KLC1</t> and KIF5B. ( A ) The co-expression of CCDC28B and KLC1 allows cdc25H yeast cells to grow at the non-permissive temperature of 37 °C only on galactose that triggers expression from the pMyr construct. Controls: MAFB-MAFB (positive); MAFB-Lamin C (negative), ColI-MAFB (negative), pSOS EV-pMyr EV (negative) and pSOS CCDC28B-pMyr EV (negative). ( B ) HA-CCDC28B is detected only in the Myc-KLC1 immunoprecipitate. Cell lysates are shown to control for protein input. Bands were cropped from the same blot which is shown in Fig. . ( C ) Immunoprecipitation of overexpressed CCDC28B in Hek293 cells with the specific single domain llama antibody (VHH) results in the co-immunoprecipitation of additional proteins. Arrows indicate gel bands analyzed by mass spectrometry. An irrelevant VHH was used as control. The gel was cut in two to avoid saturation of the VHH bands when silver-staining the upper part of the gel. ( D ) The VHH against CCDC28B was used for immunoprecipitation and specific antibodies were used to detect KIF5B, KLC1, α-tubulin and CCDC28B. Cell lysates show the corresponding proteins in the extracts used for immunoprecipitation. Bands shown were cropped from the original blot. The full-length membrane was cut and exposed to the different antibodies (see Fig. for blots and for details).
Chemically Modified Stealth Small Interfering Rnas (Sirnas) Targeting Fbxo31 And Control Sirna, supplied by Ribobio co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
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control stealth sirna oligonucleotides (12935300)  (Kaneka Corp)
90
Kaneka Corp control stealth sirna oligonucleotides (12935300)
Downregulation of cytokine and chemokine release <t>by</t> <t>ADAM8</t> knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of <t>shRNA</t> against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF- α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the mean + SD of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags ( # p < 0.05, ## p < 0.01, and ### p < 0.001).
Control Stealth Sirna Oligonucleotides (12935300), supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/control stealth sirna oligonucleotides (12935300)/product/Kaneka Corp
Average 90 stars, based on 1 article reviews
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stealth rnai negative universal control (uc) samples  (Zora Biosciences Oy)
90
Zora Biosciences Oy stealth rnai negative universal control (uc) samples
Downregulation of cytokine and chemokine release <t>by</t> <t>ADAM8</t> knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of <t>shRNA</t> against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF- α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the mean + SD of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags ( # p < 0.05, ## p < 0.01, and ### p < 0.001).
Stealth Rnai Negative Universal Control (Uc) Samples, supplied by Zora Biosciences Oy, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/stealth rnai negative universal control (uc) samples/product/Zora Biosciences Oy
Average 90 stars, based on 1 article reviews
stealth rnai negative universal control (uc) samples - by Bioz Stars, 2026-03
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stealth rnai negative control duplex  (Ribobio co)
90
Ribobio co stealth rnai negative control duplex
Downregulation of cytokine and chemokine release <t>by</t> <t>ADAM8</t> knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of <t>shRNA</t> against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF- α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the mean + SD of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags ( # p < 0.05, ## p < 0.01, and ### p < 0.001).
Stealth Rnai Negative Control Duplex, supplied by Ribobio co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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stealth rnai negative control duplex - by Bioz Stars, 2026-03
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chemically modified stealth control small interfering rnas  (Ribobio co)
90
Ribobio co chemically modified stealth control small interfering rnas
Downregulation of cytokine and chemokine release <t>by</t> <t>ADAM8</t> knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of <t>shRNA</t> against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF- α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the mean + SD of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags ( # p < 0.05, ## p < 0.01, and ### p < 0.001).
Chemically Modified Stealth Control Small Interfering Rnas, supplied by Ribobio co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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stealth rnai negative control (nc) duplexes  (Ribobio co)
90
Ribobio co stealth rnai negative control (nc) duplexes
Downregulation of cytokine and chemokine release <t>by</t> <t>ADAM8</t> knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of <t>shRNA</t> against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF- α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the mean + SD of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags ( # p < 0.05, ## p < 0.01, and ### p < 0.001).
Stealth Rnai Negative Control (Nc) Duplexes, supplied by Ribobio co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
stealth rnai negative control (nc) duplexes - by Bioz Stars, 2026-03
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Image Search Results


CCDC28B interacts with the kinesin 1 components KLC1 and KIF5B. ( A ) The co-expression of CCDC28B and KLC1 allows cdc25H yeast cells to grow at the non-permissive temperature of 37 °C only on galactose that triggers expression from the pMyr construct. Controls: MAFB-MAFB (positive); MAFB-Lamin C (negative), ColI-MAFB (negative), pSOS EV-pMyr EV (negative) and pSOS CCDC28B-pMyr EV (negative). ( B ) HA-CCDC28B is detected only in the Myc-KLC1 immunoprecipitate. Cell lysates are shown to control for protein input. Bands were cropped from the same blot which is shown in Fig. . ( C ) Immunoprecipitation of overexpressed CCDC28B in Hek293 cells with the specific single domain llama antibody (VHH) results in the co-immunoprecipitation of additional proteins. Arrows indicate gel bands analyzed by mass spectrometry. An irrelevant VHH was used as control. The gel was cut in two to avoid saturation of the VHH bands when silver-staining the upper part of the gel. ( D ) The VHH against CCDC28B was used for immunoprecipitation and specific antibodies were used to detect KIF5B, KLC1, α-tubulin and CCDC28B. Cell lysates show the corresponding proteins in the extracts used for immunoprecipitation. Bands shown were cropped from the original blot. The full-length membrane was cut and exposed to the different antibodies (see Fig. for blots and for details).

Journal: Scientific Reports

Article Title: Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B

doi: 10.1038/s41598-018-21329-6

Figure Lengend Snippet: CCDC28B interacts with the kinesin 1 components KLC1 and KIF5B. ( A ) The co-expression of CCDC28B and KLC1 allows cdc25H yeast cells to grow at the non-permissive temperature of 37 °C only on galactose that triggers expression from the pMyr construct. Controls: MAFB-MAFB (positive); MAFB-Lamin C (negative), ColI-MAFB (negative), pSOS EV-pMyr EV (negative) and pSOS CCDC28B-pMyr EV (negative). ( B ) HA-CCDC28B is detected only in the Myc-KLC1 immunoprecipitate. Cell lysates are shown to control for protein input. Bands were cropped from the same blot which is shown in Fig. . ( C ) Immunoprecipitation of overexpressed CCDC28B in Hek293 cells with the specific single domain llama antibody (VHH) results in the co-immunoprecipitation of additional proteins. Arrows indicate gel bands analyzed by mass spectrometry. An irrelevant VHH was used as control. The gel was cut in two to avoid saturation of the VHH bands when silver-staining the upper part of the gel. ( D ) The VHH against CCDC28B was used for immunoprecipitation and specific antibodies were used to detect KIF5B, KLC1, α-tubulin and CCDC28B. Cell lysates show the corresponding proteins in the extracts used for immunoprecipitation. Bands shown were cropped from the original blot. The full-length membrane was cut and exposed to the different antibodies (see Fig. for blots and for details).

Article Snippet: Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos.

Techniques: Expressing, Construct, Control, Immunoprecipitation, Mass Spectrometry, Silver Staining, Membrane

Depletion of KLC1 and KIF5B results in elongated cilia. hTERT-RPE cells were analyzed by confocal microscopy using anti-acetylated tubulin (green), anti γ-tubulin (red) and DAPI (blue) to stain cilia, basal bodies and nuclei respectively. Cilia length was measured and results are expressed as box plots. Results are representative of three independent experiments. ( A ) While knockdown of KLC1 did not affect the proportion of cilia-positive cells compared to a control stealth (hypothesis test for proportions), it did result in significantly elongated cilia (statistical test: Mann-Whitney; *** P < 0.0001). At least 100 cilia were measured per condition (143 for S.Ctrl. and 147 for S.KLC1). ( B ) Knockdown of the different KIF5s (S.KIF5A, B, C) did not affect the proportion of cilia-positive cells. Similarly to KLC1 KD, depletion of KIF5B, KIF5BC and KIF5ABC resulted in elongated cilia (122 KIF5B KD, 96 KIF5BC and 109 KIF5ABC cilia were measured and compared to 95 control). Statistical test: one-way ANOVA. Asterisks denote statistical significant differences compared to controls. *** P < 0.0001. Scale bars correspond to 10 μm.

Journal: Scientific Reports

Article Title: Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B

doi: 10.1038/s41598-018-21329-6

Figure Lengend Snippet: Depletion of KLC1 and KIF5B results in elongated cilia. hTERT-RPE cells were analyzed by confocal microscopy using anti-acetylated tubulin (green), anti γ-tubulin (red) and DAPI (blue) to stain cilia, basal bodies and nuclei respectively. Cilia length was measured and results are expressed as box plots. Results are representative of three independent experiments. ( A ) While knockdown of KLC1 did not affect the proportion of cilia-positive cells compared to a control stealth (hypothesis test for proportions), it did result in significantly elongated cilia (statistical test: Mann-Whitney; *** P < 0.0001). At least 100 cilia were measured per condition (143 for S.Ctrl. and 147 for S.KLC1). ( B ) Knockdown of the different KIF5s (S.KIF5A, B, C) did not affect the proportion of cilia-positive cells. Similarly to KLC1 KD, depletion of KIF5B, KIF5BC and KIF5ABC resulted in elongated cilia (122 KIF5B KD, 96 KIF5BC and 109 KIF5ABC cilia were measured and compared to 95 control). Statistical test: one-way ANOVA. Asterisks denote statistical significant differences compared to controls. *** P < 0.0001. Scale bars correspond to 10 μm.

Article Snippet: Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos.

Techniques: Confocal Microscopy, Staining, Knockdown, Control, MANN-WHITNEY

KLC1 and KIF5B are found at the base of cilia. Confocal microscopy analysis of hTERT-RPE cells. ( A ) Cells were transfected to overexpress Myc-tagged KLC1. In addition to cytoplasmic aggregates, a pool of Myc-KLC1 (anti-Myc, green) is found at the base of cilia (anti acetylated α-tubulin, red). ( B ) In addition to a diffuse cytoplasmic signal, the anti-KLC1 antibody shows an accumulation of endogenous protein at the base of cilia. ( C ) Similarly, endogenous KIF5B is found in the cytoplasm and also concentrated at the base of cilia. Yellow boxes mark the area that is magnified and showed in panels on the right. DAPI was used to stain nuclei. Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos. Scale bars correspond to 1 μm. ( E , F ) The level of tubulin acetylation in control and KLC1 KD cilia was quantified measuring the fluorescence intensity of the signal obtained using the acetylated α-tubulin antibody (green). The anti-ARL13 (red) signal was used to mark the entire length of the cilium. Scale bars correspond to 2 μm. Each cilium was divided in 10 segments from base to tip (see methods) and the mean intensity was computed. For each experiment (KLC1 KD and control) a ten-point intensity profile was computed by averaging the measure of all cilia in each one of the regions of interest. These profiles are shown, normalized by the measure of its first region of interest. Vertical bars plot the 95% confidence interval about the mean.

Journal: Scientific Reports

Article Title: Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B

doi: 10.1038/s41598-018-21329-6

Figure Lengend Snippet: KLC1 and KIF5B are found at the base of cilia. Confocal microscopy analysis of hTERT-RPE cells. ( A ) Cells were transfected to overexpress Myc-tagged KLC1. In addition to cytoplasmic aggregates, a pool of Myc-KLC1 (anti-Myc, green) is found at the base of cilia (anti acetylated α-tubulin, red). ( B ) In addition to a diffuse cytoplasmic signal, the anti-KLC1 antibody shows an accumulation of endogenous protein at the base of cilia. ( C ) Similarly, endogenous KIF5B is found in the cytoplasm and also concentrated at the base of cilia. Yellow boxes mark the area that is magnified and showed in panels on the right. DAPI was used to stain nuclei. Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos. Scale bars correspond to 1 μm. ( E , F ) The level of tubulin acetylation in control and KLC1 KD cilia was quantified measuring the fluorescence intensity of the signal obtained using the acetylated α-tubulin antibody (green). The anti-ARL13 (red) signal was used to mark the entire length of the cilium. Scale bars correspond to 2 μm. Each cilium was divided in 10 segments from base to tip (see methods) and the mean intensity was computed. For each experiment (KLC1 KD and control) a ten-point intensity profile was computed by averaging the measure of all cilia in each one of the regions of interest. These profiles are shown, normalized by the measure of its first region of interest. Vertical bars plot the 95% confidence interval about the mean.

Article Snippet: Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos.

Techniques: Confocal Microscopy, Transfection, Staining, Control, Fluorescence

Kinesin 1 requires CCDC28B to regulate cilia length. ( A ) The proportion of cilia-positive cells is significantly reduced upon CCDC28B KD but not KLC1 KD (hypothesis test for proportions). Co-transfection of the KLC1 stealth oligo rescues the phenotype of CCDC28B KD cells. ( B ) CCDC28B KD (118 analyzed cilia) and KLC1 KD (155 cilia measured) cells show significantly shortened and elongated cilia respectively. Cilia were of control length in cells co-transfected with both stealth oligos (115 cilia). Statistical test: one-way ANOVA; *** P < 0.001. The amount of transfected oligos per condition was maintained constant using a control stealth oligo. ( C ) CCDC28B CRISPR clone B1 was analyzed by immunofluorescence and confocal microscopy using anti-acetylated tubulin (red), anti γ-tubulin (green) and DAPI (blue) to stain cilia, basal bodies and nuclei respectively. Untreated hTERT-RPE cells were used as control and CCDC28B CRISPR clone B1 transfected with pCS2+ _CCDC28B wt was used to show rescue and specificity of the cilia phenotype. ( D , E ) Clone B1 cells were analyzed to quantify the number of total cilia-positive cells (acetylated α-tubulin signal irrespective of length) and cells bearing “long” cilia of at least 1 μm. Scale bars represent 10 μm. ( E ) Quantification shows that while the total number of cilia is similar between hTERT-RPE controls and Clone B1, the later presents significantly less cilia of at least 1 μm. Importantly, the proportion of cells bearing 1 μm cilia is rescued upon transfecting wt CCDC28B. 80 cells were analyzed for control and clone B1 cells, and 70 cells were scored for clone B1 overexpressing CCDC28B. Statistical test used: hypothesis test for proportions. Data are representative of three independent experiments. ( F ) Genetic interaction experiment using CCDC28B CRISPR clone B1. KLC1 KD and KIF5ABC KD result in a mild rescue (compared to that of KIF7) that is abolished or diminished by further CCDC28B KD for KLC1 and KIF5s respectively. KIF7 KD rescues the ciliary phenotype irrespective of the presence of CCDC28B. The experiment shown is representative of four independent experiments. The total number of cells scored for this experiment is 89 Ctrl.-Ctrl., 96 Ctrl.-CCDC28B, 91 Ctrl.-KLC1, 86 KLC1.-CCDC28B, 84 Ctrl.-KIF5ABC, 86 KIF5ABC-CCDC28B, 84 Ctrl.-KIF7, 104 KIF7.-CCDC28B. Statistical test: hypothesis test for proportions. *Indicate comparison to Clone B1 transfected with control oligos (S.CTRL-S.CTRL). ♦ Indicate comparison to Clone B1 transfected with control oligo and CCDC28B oligo (S.CTRL-S.CCDC28B). */♦ P < 0.05; ** P < 0.01; ***/♦♦♦ P < 0.001. Scale bars correspond to 10 μm.

Journal: Scientific Reports

Article Title: Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B

doi: 10.1038/s41598-018-21329-6

Figure Lengend Snippet: Kinesin 1 requires CCDC28B to regulate cilia length. ( A ) The proportion of cilia-positive cells is significantly reduced upon CCDC28B KD but not KLC1 KD (hypothesis test for proportions). Co-transfection of the KLC1 stealth oligo rescues the phenotype of CCDC28B KD cells. ( B ) CCDC28B KD (118 analyzed cilia) and KLC1 KD (155 cilia measured) cells show significantly shortened and elongated cilia respectively. Cilia were of control length in cells co-transfected with both stealth oligos (115 cilia). Statistical test: one-way ANOVA; *** P < 0.001. The amount of transfected oligos per condition was maintained constant using a control stealth oligo. ( C ) CCDC28B CRISPR clone B1 was analyzed by immunofluorescence and confocal microscopy using anti-acetylated tubulin (red), anti γ-tubulin (green) and DAPI (blue) to stain cilia, basal bodies and nuclei respectively. Untreated hTERT-RPE cells were used as control and CCDC28B CRISPR clone B1 transfected with pCS2+ _CCDC28B wt was used to show rescue and specificity of the cilia phenotype. ( D , E ) Clone B1 cells were analyzed to quantify the number of total cilia-positive cells (acetylated α-tubulin signal irrespective of length) and cells bearing “long” cilia of at least 1 μm. Scale bars represent 10 μm. ( E ) Quantification shows that while the total number of cilia is similar between hTERT-RPE controls and Clone B1, the later presents significantly less cilia of at least 1 μm. Importantly, the proportion of cells bearing 1 μm cilia is rescued upon transfecting wt CCDC28B. 80 cells were analyzed for control and clone B1 cells, and 70 cells were scored for clone B1 overexpressing CCDC28B. Statistical test used: hypothesis test for proportions. Data are representative of three independent experiments. ( F ) Genetic interaction experiment using CCDC28B CRISPR clone B1. KLC1 KD and KIF5ABC KD result in a mild rescue (compared to that of KIF7) that is abolished or diminished by further CCDC28B KD for KLC1 and KIF5s respectively. KIF7 KD rescues the ciliary phenotype irrespective of the presence of CCDC28B. The experiment shown is representative of four independent experiments. The total number of cells scored for this experiment is 89 Ctrl.-Ctrl., 96 Ctrl.-CCDC28B, 91 Ctrl.-KLC1, 86 KLC1.-CCDC28B, 84 Ctrl.-KIF5ABC, 86 KIF5ABC-CCDC28B, 84 Ctrl.-KIF7, 104 KIF7.-CCDC28B. Statistical test: hypothesis test for proportions. *Indicate comparison to Clone B1 transfected with control oligos (S.CTRL-S.CTRL). ♦ Indicate comparison to Clone B1 transfected with control oligo and CCDC28B oligo (S.CTRL-S.CCDC28B). */♦ P < 0.05; ** P < 0.01; ***/♦♦♦ P < 0.001. Scale bars correspond to 10 μm.

Article Snippet: Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos.

Techniques: Cotransfection, Control, Transfection, CRISPR, Immunofluorescence, Confocal Microscopy, Staining, Comparison

KLC1 plays a role regulating the sub-cellular distribution of CCDC28B. ( A ) Confocal microscopy analysis of endogenous CCDC28B (green) in hTERT-RPE cells showing localization of the protein in the cytoplasm, pericentriolar region/basal body (arrows illustrate examples in a ciliated and a non-ciliated cell, higher magnification in yellow box) and the nucleus (circle). Basal bodies and cilia axoneme were stained with anti-γ- and anti-acetylated α-tubulin respectively (red). ( B ) CCDC28B (green) signal is increased in the nucleus upon KLC1 KD (S.KLC1; lower panels) compared to control cells (S.CTRL; upper panels). In both ( A , B ), DAPI was used to stain nuclei. Scale bars correspond to 10 μm. ( C ) Sub-cellular fractionation assay using hTERT-RPE cells transfected with a Myc-CCDC28B expressing plasmid together with stealth control (S.CTRL) or stealth KLC1 (S.KLC1). CCDC28B is present in both the cytosolic and nuclear fractions in control cells and accumulates in the nuclear fraction in KLC1 KD cells. The membrane was cut at the 35 KDa ladder band. The blot incubated with the α-Myc to visualize CCDC28B was stripped and probed with α-Histone. The graph shows the nuclear/total (nuclear + cytoplasmic) ratio obtained by quantifying the western blot bands by densitometry. α-tubulin was used to normalize the nuclear intensity of CCDC28B and compensate for the cytosolic contamination in the nuclear fraction.

Journal: Scientific Reports

Article Title: Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B

doi: 10.1038/s41598-018-21329-6

Figure Lengend Snippet: KLC1 plays a role regulating the sub-cellular distribution of CCDC28B. ( A ) Confocal microscopy analysis of endogenous CCDC28B (green) in hTERT-RPE cells showing localization of the protein in the cytoplasm, pericentriolar region/basal body (arrows illustrate examples in a ciliated and a non-ciliated cell, higher magnification in yellow box) and the nucleus (circle). Basal bodies and cilia axoneme were stained with anti-γ- and anti-acetylated α-tubulin respectively (red). ( B ) CCDC28B (green) signal is increased in the nucleus upon KLC1 KD (S.KLC1; lower panels) compared to control cells (S.CTRL; upper panels). In both ( A , B ), DAPI was used to stain nuclei. Scale bars correspond to 10 μm. ( C ) Sub-cellular fractionation assay using hTERT-RPE cells transfected with a Myc-CCDC28B expressing plasmid together with stealth control (S.CTRL) or stealth KLC1 (S.KLC1). CCDC28B is present in both the cytosolic and nuclear fractions in control cells and accumulates in the nuclear fraction in KLC1 KD cells. The membrane was cut at the 35 KDa ladder band. The blot incubated with the α-Myc to visualize CCDC28B was stripped and probed with α-Histone. The graph shows the nuclear/total (nuclear + cytoplasmic) ratio obtained by quantifying the western blot bands by densitometry. α-tubulin was used to normalize the nuclear intensity of CCDC28B and compensate for the cytosolic contamination in the nuclear fraction.

Article Snippet: Scale bars correspond to 10 μm. ( D ) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos.

Techniques: Confocal Microscopy, Staining, Control, Cell Fractionation, Transfection, Expressing, Plasmid Preparation, Membrane, Incubation, Western Blot

Downregulation of cytokine and chemokine release by ADAM8 knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of shRNA against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF- α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the mean + SD of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags ( # p < 0.05, ## p < 0.01, and ### p < 0.001).

Journal: Mediators of Inflammation

Article Title: Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

doi: 10.1155/2021/6665028

Figure Lengend Snippet: Downregulation of cytokine and chemokine release by ADAM8 knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of shRNA against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF- α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the mean + SD of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags ( # p < 0.05, ## p < 0.01, and ### p < 0.001).

Article Snippet: Murine Hepa1-6 hepatoma cells were transfected with two different ADAM8 stealth siRNA nucleotides (83234478) or control stealth siRNA oligonucleotides (12935300) (Eurogentec, Liège, Belgium), using Lipofectamine RNAiMAX (Invitrogen, Germany) according to the manufacturer's instructions.

Techniques: Transduction, shRNA, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Stimulation