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Impact of <t>Alkbh1</t> knockdown on cell fate and tRNA cleavage after stress. A: Annexin V FACS analysis (left graph) and SYBR gold staining following Alkbh1 KD and arsenite stress (400 µM for 4 hours). Alkbh1 KD reduced the levels of apoptotic and necrotic cells and rescued tRNA from cleavage. B: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and antimycin stress (150 µg/ml for 4 hours). Alkbh1 KD had no impact on cell fate or tRNA cleavage following antimycin stress. (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation. FACS data were presented as ratio to unstressed (control) cells). C: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and OGD-R stress. Alkbh1 KD also had no impact on cell death ratio following OGD-R and no apparent impact on tRNA cleavage. Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane.

Rat Alkbh1 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rat alkbh1 sirna - by Bioz Stars, 2024-09

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1) Product Images from "The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation"

Article Title: The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation

Journal: RNA Biology

doi: 10.1080/15476286.2020.1779492

Impact of Alkbh1 knockdown on cell fate and tRNA cleavage after stress. A: Annexin V FACS analysis (left graph) and SYBR gold staining following Alkbh1 KD and arsenite stress (400 µM for 4 hours). Alkbh1 KD reduced the levels of apoptotic and necrotic cells and rescued tRNA from cleavage. B: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and antimycin stress (150 µg/ml for 4 hours). Alkbh1 KD had no impact on cell fate or tRNA cleavage following antimycin stress. (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation. FACS data were presented as ratio to unstressed (control) cells). C: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and OGD-R stress. Alkbh1 KD also had no impact on cell death ratio following OGD-R and no apparent impact on tRNA cleavage. Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane.

Figure Legend Snippet: Impact of Alkbh1 knockdown on cell fate and tRNA cleavage after stress. A: Annexin V FACS analysis (left graph) and SYBR gold staining following Alkbh1 KD and arsenite stress (400 µM for 4 hours). Alkbh1 KD reduced the levels of apoptotic and necrotic cells and rescued tRNA from cleavage. B: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and antimycin stress (150 µg/ml for 4 hours). Alkbh1 KD had no impact on cell fate or tRNA cleavage following antimycin stress. (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation. FACS data were presented as ratio to unstressed (control) cells). C: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and OGD-R stress. Alkbh1 KD also had no impact on cell death ratio following OGD-R and no apparent impact on tRNA cleavage. Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane.

Techniques Used: Staining

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Thermo Fisher rat alkbh1 sirna

A: B35 cells were exposed to increasing concentrations of arsenite (As), antimycin A for 6 hours or to 16 hours of oxygen-glucose deprivation (OGD) and OGD with 1 hour of reperfusion (OGD+R). SYBR gold staining was performed following electrophoresis of 2µg RNA per well and showed a correlation between the stress level and tRNA cleavage in all stresses [Cropped membranes are shown as a whole in supplementary figure 1]. B: Immunonorthern blotting (INB) using anti-m 1 A antibody showed that while following arsenite stress 3’tiRNA fragments harboring m 1 A were detected, antimycin stress showed much lower levels of these fragments. OGD-R showed no m 1 A containing 3’tiRNA fragments. Both antimycin and OGD-R caused reduction of m 1 A levels in full length tRNA as well, something that was not observed to that extent following arsenite stress [Cropped membranes are shown as a whole in supplementary figure 2]. C: Western blotting analysis to confirm the effectiveness of <t>Alkbh1</t> knockdown following siRNA. Alkbh1 protein levels were reduced to about 40-50% of the baseline levels as compared to scramble (negative) siRNA.

rat alkbh1 sirna - by Bioz Stars, 2024-09

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1) Product Images from "The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation"

Article Title: The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation

Journal: bioRxiv

doi: 10.1101/2020.01.31.928234

Figure Legend Snippet: A: B35 cells were exposed to increasing concentrations of arsenite (As), antimycin A for 6 hours or to 16 hours of oxygen-glucose deprivation (OGD) and OGD with 1 hour of reperfusion (OGD+R). SYBR gold staining was performed following electrophoresis of 2µg RNA per well and showed a correlation between the stress level and tRNA cleavage in all stresses [Cropped membranes are shown as a whole in supplementary figure 1]. B: Immunonorthern blotting (INB) using anti-m 1 A antibody showed that while following arsenite stress 3’tiRNA fragments harboring m 1 A were detected, antimycin stress showed much lower levels of these fragments. OGD-R showed no m 1 A containing 3’tiRNA fragments. Both antimycin and OGD-R caused reduction of m 1 A levels in full length tRNA as well, something that was not observed to that extent following arsenite stress [Cropped membranes are shown as a whole in supplementary figure 2]. C: Western blotting analysis to confirm the effectiveness of Alkbh1 knockdown following siRNA. Alkbh1 protein levels were reduced to about 40-50% of the baseline levels as compared to scramble (negative) siRNA.

Techniques Used: Staining, Electrophoresis, Western Blot

A: Annexin V FACS analysis (left graph) and SYBR gold staining following Alkbh1 KD and arsenite stress (400µM for 4 hours). Alkbh1 KD reduced the levels of apoptotic and necrotic cells and rescued tRNA from cleavage. B: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and antimycin stress (150µg/ml for 4 hours). Alkbh1 KD had no impact on cell fate or tRNA cleavage following antimycin stress. (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation. FACS data were presented as ratio to unstressed (control) cells). C: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and OGD-R stress. Alkbh1 KD also had no impact on cell death ratio following OGD-R and no apparent impact on tRNA cleavage. Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane.

Figure Legend Snippet: A: Annexin V FACS analysis (left graph) and SYBR gold staining following Alkbh1 KD and arsenite stress (400µM for 4 hours). Alkbh1 KD reduced the levels of apoptotic and necrotic cells and rescued tRNA from cleavage. B: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and antimycin stress (150µg/ml for 4 hours). Alkbh1 KD had no impact on cell fate or tRNA cleavage following antimycin stress. (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation. FACS data were presented as ratio to unstressed (control) cells). C: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and OGD-R stress. Alkbh1 KD also had no impact on cell death ratio following OGD-R and no apparent impact on tRNA cleavage. Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane.

Techniques Used: Staining

A: Cytosolic tRNA cleavage before and after Alkbh1 KD following 4 hours of arsenite (As; 400µM) or antimycin (A.M; 150µg/ml) stresses. Northern blotting performed with 1µg total RNA per lane. Leu AAG 5’tiRNA cleavage was significantly reduced after Alkbh1 KD following As stress, with little to no effect on its cleavage following A.M stress. There was a notable but unexplained reduction in the full length tRNA levels following KD and A.M stress. Moreover, an extra band appeared after As cleavage and KD just below the full length tRNA (similar to one observed with A.M stress) which cannot be explained at the present time (black arrow: 5’tiRNA, white arrow: full length tRNA, Red arrow: large 5’tiRNA fragments). Ala TGC was cleaved into several 5’tiRNAs of varying lengths especially after A.M stress (Blue and black arrows). KD of Alkbh1 reduced the cleavage after As but not A.M stress. Again, the full length tRNA levels were reduced with KD following A.M stress and there is an apparent shift in the tRNA size between A.M and As and control samples. This may indicate partial cleavage of tRNA (white arrow). Gly GCC was cleaved into 2 distinct 5’ fragments, one at the usual size of the 5’tiRNA (black arrow), and another large fragment around 50∼60 nt in length (red arrow). Alkbh1 KD reduced Gly GCC cleavage after As stress, while after A.M stress the larger fragments showed a decrease in signal while the canonical fragments (black arrow) were not significantly changed. Glu CTC also showed cleavage into 2 5’tiRNA fragments. Alkbh1 KD impacted the cleavage after As stress only. Lys CTT was also cleaved into 2 5’tiRNA fragments. Alkbh1 KD reduced the cleavage following As and A.M stresses. Arg CCT cleavage was apparent in the form of large 5’tiRNA fragment after A.M stress only, and this fragment was minimally affected by Alkbh1 KD (although this effect can be negligible). Met CAT and Gln CTG did not exhibit 5’tiRNA fragments after stress, but there was a reduction in their full length tRNA levels (white arrows). Alkbh1 had no apparent impact on these 2 tRNAs. B: Mitochondrial tRNA cleavage before and after Alkbh1 KD following 4 hours of arsenite (As; 400µM) or antimycin (A.M; 150µg/ml) stresses. Northern blotting performed with 4µg total RNA per lane. Mt-Tyr GTA was cleaved into several fragments after As and A.M cleavage, with more robust cleavage after A.M stress. Alkbh1 KD reduced mt-Tyr GTA cleavage after As but not A.M stress. Notably, the largest of the 5’tiRNA fragments (red arrow) was reduced with Alkbh1 KD following A.M stress. Moreover, full length tRNA levels (white arrow) were reduced after KD. mt-Asn GTT was cleaved into a larger 5’tiRNA fragment (red arrow) which was reduced with Alkbh1 KD following As and A.M stresses. mt-Met CAT was cleaved into a large 5’tiRNA fragment as well apparent only after A.M stress. Alkbh1 KD minimally reduced the signal of the 5’tiRNA-mt-Met CAT . mt-Gly TCC and mt-Ala TGC were not apparently cleaved and showed no impact of Alkbh1 on their levels. [Membranes were not cropped]

Figure Legend Snippet: A: Cytosolic tRNA cleavage before and after Alkbh1 KD following 4 hours of arsenite (As; 400µM) or antimycin (A.M; 150µg/ml) stresses. Northern blotting performed with 1µg total RNA per lane. Leu AAG 5’tiRNA cleavage was significantly reduced after Alkbh1 KD following As stress, with little to no effect on its cleavage following A.M stress. There was a notable but unexplained reduction in the full length tRNA levels following KD and A.M stress. Moreover, an extra band appeared after As cleavage and KD just below the full length tRNA (similar to one observed with A.M stress) which cannot be explained at the present time (black arrow: 5’tiRNA, white arrow: full length tRNA, Red arrow: large 5’tiRNA fragments). Ala TGC was cleaved into several 5’tiRNAs of varying lengths especially after A.M stress (Blue and black arrows). KD of Alkbh1 reduced the cleavage after As but not A.M stress. Again, the full length tRNA levels were reduced with KD following A.M stress and there is an apparent shift in the tRNA size between A.M and As and control samples. This may indicate partial cleavage of tRNA (white arrow). Gly GCC was cleaved into 2 distinct 5’ fragments, one at the usual size of the 5’tiRNA (black arrow), and another large fragment around 50∼60 nt in length (red arrow). Alkbh1 KD reduced Gly GCC cleavage after As stress, while after A.M stress the larger fragments showed a decrease in signal while the canonical fragments (black arrow) were not significantly changed. Glu CTC also showed cleavage into 2 5’tiRNA fragments. Alkbh1 KD impacted the cleavage after As stress only. Lys CTT was also cleaved into 2 5’tiRNA fragments. Alkbh1 KD reduced the cleavage following As and A.M stresses. Arg CCT cleavage was apparent in the form of large 5’tiRNA fragment after A.M stress only, and this fragment was minimally affected by Alkbh1 KD (although this effect can be negligible). Met CAT and Gln CTG did not exhibit 5’tiRNA fragments after stress, but there was a reduction in their full length tRNA levels (white arrows). Alkbh1 had no apparent impact on these 2 tRNAs. B: Mitochondrial tRNA cleavage before and after Alkbh1 KD following 4 hours of arsenite (As; 400µM) or antimycin (A.M; 150µg/ml) stresses. Northern blotting performed with 4µg total RNA per lane. Mt-Tyr GTA was cleaved into several fragments after As and A.M cleavage, with more robust cleavage after A.M stress. Alkbh1 KD reduced mt-Tyr GTA cleavage after As but not A.M stress. Notably, the largest of the 5’tiRNA fragments (red arrow) was reduced with Alkbh1 KD following A.M stress. Moreover, full length tRNA levels (white arrow) were reduced after KD. mt-Asn GTT was cleaved into a larger 5’tiRNA fragment (red arrow) which was reduced with Alkbh1 KD following As and A.M stresses. mt-Met CAT was cleaved into a large 5’tiRNA fragment as well apparent only after A.M stress. Alkbh1 KD minimally reduced the signal of the 5’tiRNA-mt-Met CAT . mt-Gly TCC and mt-Ala TGC were not apparently cleaved and showed no impact of Alkbh1 on their levels. [Membranes were not cropped]

Techniques Used: Northern Blot

A: Western blotting confirmation of Alkbh1 overexpression. Lenti-encoded Alkbh1 with GFP-attached sequence resulted in a larger protein (black arrow) than the endogenous Alkbh1 (blue arrow). B: Arsenite stress (4 hours, 1mM) resulted in more cell death in B35 ALK than in B35 Mock cells as revealed by Annexin V FACS assay (left graphs). This effect was reflected in the form of increased gross tRNA cleavage and tiRNA generation observed on SYBR gold staining (Right). C: Antimycin stress (4 hours, 200µg/ml) resulted in equal levels of cell death in B35 Mock and B35 ALK (left: Annexin V FACS analysis) and equal levels of tRNA cleavage (Right: SYBR gold staining). (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation). Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane. FACS data were presented as fold-change unstressed cells.

Figure Legend Snippet: A: Western blotting confirmation of Alkbh1 overexpression. Lenti-encoded Alkbh1 with GFP-attached sequence resulted in a larger protein (black arrow) than the endogenous Alkbh1 (blue arrow). B: Arsenite stress (4 hours, 1mM) resulted in more cell death in B35 ALK than in B35 Mock cells as revealed by Annexin V FACS assay (left graphs). This effect was reflected in the form of increased gross tRNA cleavage and tiRNA generation observed on SYBR gold staining (Right). C: Antimycin stress (4 hours, 200µg/ml) resulted in equal levels of cell death in B35 Mock and B35 ALK (left: Annexin V FACS analysis) and equal levels of tRNA cleavage (Right: SYBR gold staining). (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation). Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane. FACS data were presented as fold-change unstressed cells.

Techniques Used: Western Blot, Over Expression, Sequencing, Staining

A: Cytosolic tRNA cleavage in B35 Mock and B35 ALK cells after 4 hours of As (1mM) or A.M (200µg/ml) stresses. Northern blotting performed with 1µg total RNA per lane, Alkbh1 overexpression led to a general pattern of increased tRNA cleavage. Leu AAG , Ala TGC , Gly GCC , Glu CTC , Lys CTT and Arg CCT showed increased cleavage in their 5’tiRNA fragments (whether canonical or larger fragments) in a pattern opposite to what was observed with Alkbh1 KD . Moreover, Met CAT and Gln CTG , which did not exhibit apparent cleavage with stress and siRNA KD, were cleaved with Alkbh1 overexpression into large 5’tiRNA fragments after A.M stress (red arrows). B: Mitochondrial tRNA cleavage in B35 Mock and B35 ALK cells after As and A.M stresses. Northern blotting performed with 4µg total RNA per lane. mt-Tyr GTA cleavage after As in B35 ALK cells was not impacted in an apparent manner, however, after A.M stress, the larger fragments (red arrow), which were reduced after siRNA KD in , did exhibit some increase in its signal. mt-Met CAT cleavage after A.M stress in B35 ALK cells was greatly enhanced, with the larger fragment (red arrow) being clearly visible and the appearance of 2 other fragments that were not observable prior to Alkbh1 overexpression (black arrows). mt-Asn GTT showed an enhanced cleavage after Alkbh1 overexpression (red arrow), with the observation of faint signal of a smaller 5’tiRNA fragment not observable prior to overexpression (black arrow). mt-Gly TCC , mt-Arg TCG and mt-Ala TGC that were not cleaved prior to Alkbh1 overexpression showed no impact of Alkbh1 on their levels.

Figure Legend Snippet: A: Cytosolic tRNA cleavage in B35 Mock and B35 ALK cells after 4 hours of As (1mM) or A.M (200µg/ml) stresses. Northern blotting performed with 1µg total RNA per lane, Alkbh1 overexpression led to a general pattern of increased tRNA cleavage. Leu AAG , Ala TGC , Gly GCC , Glu CTC , Lys CTT and Arg CCT showed increased cleavage in their 5’tiRNA fragments (whether canonical or larger fragments) in a pattern opposite to what was observed with Alkbh1 KD . Moreover, Met CAT and Gln CTG , which did not exhibit apparent cleavage with stress and siRNA KD, were cleaved with Alkbh1 overexpression into large 5’tiRNA fragments after A.M stress (red arrows). B: Mitochondrial tRNA cleavage in B35 Mock and B35 ALK cells after As and A.M stresses. Northern blotting performed with 4µg total RNA per lane. mt-Tyr GTA cleavage after As in B35 ALK cells was not impacted in an apparent manner, however, after A.M stress, the larger fragments (red arrow), which were reduced after siRNA KD in , did exhibit some increase in its signal. mt-Met CAT cleavage after A.M stress in B35 ALK cells was greatly enhanced, with the larger fragment (red arrow) being clearly visible and the appearance of 2 other fragments that were not observable prior to Alkbh1 overexpression (black arrows). mt-Asn GTT showed an enhanced cleavage after Alkbh1 overexpression (red arrow), with the observation of faint signal of a smaller 5’tiRNA fragment not observable prior to overexpression (black arrow). mt-Gly TCC , mt-Arg TCG and mt-Ala TGC that were not cleaved prior to Alkbh1 overexpression showed no impact of Alkbh1 on their levels.

Techniques Used: Northern Blot, Over Expression

Immunonorthern blotting (INB) with anti-m 1 A antibody following stress and Alkbh1 siRNA KD ( A and B ) or Alkbh1 overexpression ( C ). 4µg total RNA were loaded per lane to enhance the resulting signal. A: As and A.M stress both resulted in tRNA cleavage as observed with the existence of m 1 A-harboring 3’tiRNA fragments (black arrow). As and A.M stresses both led to a demethylation effect on tRNA (yellow arrow), however, As stress resulted in a moderate demethylation as opposed to the significant demethylation observed with A.M. Alkbh1 KD resulted in decrease tRNA cleavage and recovery of tRNA m 1 A methylation status after As stress, while after A.M stress there was no impact of Alkbh1 KD on tRNA cleavage and tRNA was further demethylated in a paradoxical manner. B: Alkbh1 KD following OGD-R stress resulted in decreases m 1 A signal in full length tRNA indicating a paradoxical demethylating effect. C: INB after As (1mM) and A.M (200µg/ml) stress exposure of B35 Mock and B35 ALK for 4 hours. Alkbh1 overexpression resulted in modest demethylating effect on tRNA after As stress and a significant demethylation after A.M (yellow arrow). Alkbh1 overexpression also led to increase in m 1 A-harboring tiRNAs after As stress and a paradoxical reduction of such fragments after A.M stress indicating that the demethylation also impacts the resulting tiRNAs.

Figure Legend Snippet: Immunonorthern blotting (INB) with anti-m 1 A antibody following stress and Alkbh1 siRNA KD ( A and B ) or Alkbh1 overexpression ( C ). 4µg total RNA were loaded per lane to enhance the resulting signal. A: As and A.M stress both resulted in tRNA cleavage as observed with the existence of m 1 A-harboring 3’tiRNA fragments (black arrow). As and A.M stresses both led to a demethylation effect on tRNA (yellow arrow), however, As stress resulted in a moderate demethylation as opposed to the significant demethylation observed with A.M. Alkbh1 KD resulted in decrease tRNA cleavage and recovery of tRNA m 1 A methylation status after As stress, while after A.M stress there was no impact of Alkbh1 KD on tRNA cleavage and tRNA was further demethylated in a paradoxical manner. B: Alkbh1 KD following OGD-R stress resulted in decreases m 1 A signal in full length tRNA indicating a paradoxical demethylating effect. C: INB after As (1mM) and A.M (200µg/ml) stress exposure of B35 Mock and B35 ALK for 4 hours. Alkbh1 overexpression resulted in modest demethylating effect on tRNA after As stress and a significant demethylation after A.M (yellow arrow). Alkbh1 overexpression also led to increase in m 1 A-harboring tiRNAs after As stress and a paradoxical reduction of such fragments after A.M stress indicating that the demethylation also impacts the resulting tiRNAs.

Techniques Used: Over Expression, Methylation

rat alkbh1 sirna (Thermo Fisher)

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rat alkbh1 sirna (Thermo Fisher)

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1) Product Images from "The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation"

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